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BSC 3403
Final Exam
| Term | Definition |
|---|---|
| Molecular biology | the study of DNA and proteins and their interactions, and the molecular information transfer within a cell. |
| Recombinant DNA technology | the process of taking a gene from one source (such as human DNA) and joining it with another piece of DNA (such as a bacterial plasmid) and inserting that DNA into bacteria for further study and manipulation. |
| DNA (deoxyribonucleic acid) | contains two strands of nucleic acids antiparallel to one another; AT; GC; negatively charged phosphate backbone 5'-3'; nucleotide DNA bases are held together by hydrogen bonds and base stacking. |
| Probes | can be used to determine the presence or absence of an allele, which can indicate a predisposition to a specific disease; are useful in generating pedigrees, paternity testing, and forensics. |
| Proteins | consist of amino acids linked by a peptide bond between the carboxyl terminus of one amino acid and the amino terminus of the next; synthesis by the ribosome is from the N- to C-terminus; 22 amino acids used |
| Primary structure | a protein’s amino acid sequence; |
| Secondary structure | alpha helices and beta sheets are the most common; |
| Tertiary structure | further folding into a complex, 3D structure; |
| Quaternary structure | multiple polypeptide chains interacting with one another; best represented by the molecular surface (solvent-accessible surface representation). |
| Central dogma of molecular biology | DNA is copied by DNA polymerase during the S phase of cell division, and mRNA is transcribed from DNA by RNA polymerase. The ribosome reads this RNA message and translates it into protein. |
| Research laboratories must | follow the rules set forth by the Occupational Safety and Health Administration (OSHA), which states that employers must inform employees of potential hazards, and that every hazardous substance has a MSDS |
| Material Safety Data Sheet (MSDS) | states a chemical’s physical and chemical properties and any associated physical hazards, health hazards, the primary route of entry, exposure limits, and whether or not it is toxic, carcinogenic, flammable, or radioactive; |
| BSL-1 laboratories | have minimal potential hazards to laboratory personnel. |
| BSL-2 laboratories | work with organisms that are of moderate potential hazard and researchers must be trained with proper safe handling, protective equipment must be worn, and waste must be disposed of properly. |
| BSL-3 laboratories | work with pathogenic organisms that can cause serious disease. Labs must be properly ventilated, access is restricted, and special protective equipment must be utilized. |
| BSL-4 laboratories | work with dangerous pathogens that cause severe to fatal disease for which vaccines or treatments are not available. |
| Centrifuge | a device that spins samples held in tubes in a rotor at very high speeds and at many times the force of gravity (g). |
| Preparative centrifuges | used to prepare samples for further use; separate molecules and can help to isolate or purify a sample; small bench top (13,000 rpm, 7000g); Large-capacity refrigerated (6000 rpm, 6500g); High-speed refrigerated(25,000rpm, 60,000g); balance within .25g |
| Analytical centrifuges | here the sedimentation characteristics of pure biological macromolecules and molecular structures can be analyzed; ultracentrifuges (80,000 rpm, 600,000g); operate under vacuum and laser to watch balance; balance within 0.1g |
| Fixed-angle rotors | allow for excellent pelleting cells from media or precipitated DNA from a solution. |
| Swinging-bucket rotors | do not create pellets (any sedimented material will be spread across the bottom of the tube) and are best for sample analysis or running cesium chloride or sucrose density gradients in an ultracentrifuge. |
| Clear bottles | allow for better visibility, but are less resistant to chemicals and can weaken and crack over time. |
| Opaque bottles | are used by researchers most often because of the disadvantages of clear bottles. |
| Electromagnetic radiation | a type of energy (a self-propagating wave) that is transmitted through space at enormous velocities and includes visible light, heat gamma-rays, X-rays, UV light, infrared light, microwaves, and radio waves. |
| Spectroscopy | the study of this [electromagnetic] radiation interacting with matter, and can be used to analyze molecular structures or dynamics through absorbance, emission, and scattering. |
| Spectrometry | the measurement of these interactions and allows one to determine molecular structure or the concentrations of solutions and can be measured wither qualitatively or quantitatively. |
| Absorbance | a characteristic of a substance that can be used to quantitatively determine the amount of substance in a solution. |
| Colorimetry | the conversion of a molecule to a colored compound by a chromogenic (color-forming) reaction; preferred over spectro. because this allows the measurement of the sample at other wavelengths that won’t be affected by DNA absorbance if the sample is impure |
| Spectrophotometers | devices used to measure the intensity of a specific wavelength of light as it passes through a sample. |
| 260 nm | DNA absorption/concentration (special cuvettes required) |
| 280 nm | Protein absorption/concentration (special cuvettes required) |
| 340 nm | GSH-CDNB in the CDNB Assay, NADH (enzyme assays) |
| 560 nm | BCA Assay (protein concentration) |
| 595 nm | Bradford Assay (protein concentration) |
| 600 nm | Bacterial growth in LB media |
| 750 nm | Lowry/DC Protein Assay (protein concentration) |
| plate readers | can quickly read microliter volumes of dozens or hundreds of samples from a single plate instead of one sample at a time. |
| Absorbance (A)= | 2-log(%) = -logT= εcl |
| Transmittance (T)= | 10^(-A)=I/I_0 |
| If the ratio of A260:A280 > 1.5, | the sample is considered pure with few protein containments. |
| Nanodrop | can measure microliter quantities of a DNA sample and determine the concentration, 260/280 ratio, and level of organic containments (A230) that may have been used during DNA purification. |
| The most accurate technique for determining protein concentration is | amino acid hydrolysis followed by chromatography but is unrealistic for routine protein concentration determination as it must be sent to a specially equipped laboratory and the costs are high (hundreds of dollars per sample) and a 2-5 day turnaround |
| Bradford Assay (Colorimetric protein quantification) | uses a dye called Coomassie Brilliant Blue that changes color from red (465 nm) to blue (595 nm) as the dye (VW) binds to proteins; nonlinear over wide range; 2x the variability than others; some compounds are incompatible in it; must be diluted; stnd crv |
| Biuret Method (Colorimetric protein quantification) | simplest method; involves copper (II) binding to the peptide nitrogen of proteins, which absorbs light at 550 nm; not very sensitive and does not work well in many common buffers, such as Tris |
| Lowry Method (Colorimetric protein quantification) | uses copper binding to peptide bonds; a folin reagent is added which becomes reduced by the Cu+ and turns blue, which is measurable at 750 nm; cheap and effective; low variability; can be detergent compatible; must be diluted; standard curve; |
| BCA Method (Colorimetric protein quantification) | similar to the Lowry Method but instead of the folin regent, BCA reagent is sued and measured at 560 nm; fast, the reagents are stable, and it has very low (~15%) variation between different proteins. |
| standard curve (Bradford, Lowry, and BCA Assays) | should always be used with a standard curve, which allows one to accurately correlate absorbance in the spectrophotometer to protein concentration; BSA can be used to create a standard curve; can be used to determine the [protein] of an unknown protien |
| Enzymes | are proteins or RNA that are essential to cell function by increasing the rate of a reaction; highly specific with respect to the substrates, single func. group can alter inter; very tightly regulated by cells; named based upon the reaction they catalyze; |
| Free energy (ΔG) | is a measure of the difference in energy between the substrates and products, and the change in free energy dictates whether a reaction will be energetically favorable. |
| A negative free energy represents | a thermodynamically favorable, spontaneous reaction (exothermic). |
| A positive free energy represents | a thermodynamically unfavorable reaction that requires an input of energy (endothermic). |
| Active site | is often a cleft or crevice on the surface of the enzyme which enhances the binding of the substrate, which interact with the active site via multiple weak forces; may require cofactors (metal) or coenzymes (organic molecules) to help reaction |
| the Lock and Key Model | proposed by Emil Fischer in 1894, the shape of the substrate and the active site of the enzyme fit together like a key into its lock (complementary); does not explain transition state stabilization. |
| Induced Fit Model | proposed by Daniel E. Koshland in 1958, the binding of substrate induces conformational changes in the enzyme, which bring the substrates together, and stabilizes the transition state. |
| Enzyme kinetics | the study of an enzyme’s rate, or how much substrate is converted to product in a given amount of time. |
| Competitive inhibitors | bind to the same site on the enzyme that the substrate bind (the active site), this competing with substrates; affected by substrate concentration |
| Noncompetitive inhibitors | bind to a second site on the enzyme, causing conformational changes in the enzyme that prevent enzyme-substrate interactions; NOT affected by substrate concentration |
| enzyme assay | can be used to determine an enzyme’s rate. |
| kinase assay | Mix a protein kinase (or a lysate) with a substrate that can be phosphorylated and Radiolabeled (32P) ATP; Run sample on gel, develop, a band indicates radioactivity and therefore phosphorylation; Can be used to study signaling pathways or test inhibitors |
| zero order reaction | if the change in concentration of substrate produces no effect |
| 1st order reaction | if doubling the concentration of substrate causes the rate to double |
| 2nd order reaction | if doubling the concentration of substrate causes the rate to quadruple |
| Solute | the molecule being dissolved, such as salt, protein, or DNA, and is usually measured in grams or moles. |
| Solvent | the solution responsible for dissolving the solute, and it is usually water and measured in liters. |
| To prepare solutions of known molarity | Amount needed (in grams)= M_w × desired concentration (M) × volume needed (L) |
| Dilutions can be calculated using the following equation | (M1×V1)= (M2×V2) |
| Buffer solutions | usually consist of a mixture of a weak acid or base and its salt; have a range in which their resistance to changes in pH are at a maximum, based on the pKa |
| lab reports | are generally used as a teaching tool to promote scientific thought, data analysis, and scientific writing |
| Seminars, or journal clubs | usually held at the program or department level, and usually involves graduate students and postdocs;practice reading a paper, analyzing the data, and presenting it to others and be brought up to date on a specific area of research |
| Invited seminar series | involve a department or entire campus, and allows for a large group of scientists to see current research from an internal or external researcher, who may given be given an honorarium (payment). |
| Conferences and symposiums are | gatherings of experts in a field, sometimes once a year that features posters and invited lecturers; allow scientists to present their research to colleagues, learn new techniques, and discuss new theories and the direction of the field. |
| A poster session | can be on-campus to display one’s research to other faculty and fellow students at a campus symposium, or at a conference where invited lecturers are presenting data |
| R01 (a Research Project Grant) | can provide hundreds of thousands or millions of dollars to a laboratory and is funded for 3-5 years; New investigators (scientists earlier in their careers) are given special consideration. |
| The top scientific journals are | Nature, Science, the New England Journal of Medicine, and Cell, as determined by their impact factor, or total number of citations for each article in the following two years by other research publications. |
| 1st author in the authorship order | the graduate student or postdoc assigned to the project by the principle investigator |
| 2nd author in the authorship order | usually a close collaborator on the project |
| middle authors in the authorship order | laboratory technicians or other research scientists who contributed to the paper |
| penultimate author in the authorship order | often a supervising technical collaborator (such as another PI) |
| final author in the authorship order | the principle investigator of the laboratory and usually the corresponding author of the paper. |
| An independent variable | is that which is manipulated (time, initial amounts, etc.). |
| A dependent variable | changes in response to the independent variable and is observed and measured. |
| A control | acts as a baseline from which to measure changes in the dependent variable. |
| Positive controls | are used as the “normal” test and should produce expected, measurable results; These ensure that the experiment worked as designed. |
| Negative controls | should not be observed, and if they are seen it indicates that the sample or reagents are contaminated. |
| Experimenter bias | involves one subconsciously biasing their data (even computer software can be biased in its data selection or analysis). |
| To prevent bias | scientists use good controls, statistically analyze data, report all data (both good and bad) and any manipulations thereof, undergo the peer review process and experiment duplication, use double-blind experiments, and hold to scientific rigor. |
| scientific rigor | ensures the data and conclusions are consistent across all experiments, and helps to prevent experimenter bias. |
| peer review process | Most journals require three anonymous peer reviewers; Generally, two of three reviewers must come back favorable for publication; disadvantage to peer review is that the process can be tedious |
| synthesizing a protein from amino acids using organic chemistry is | limited to ~50-70 amino acids (smaller than most proteins), but is useful for obtaining short peptides for research purposes; Another issue that can arise is that proteins may not always fold properly if not expressed in a cell. |
| in vitro (cell free) expression system involves | a ribosome and the mRNA of interest; very fast and produces pure protein that does not need to undergo purification, but is currently quite expensive compared to other techniques, and the amount of protein produced is very low (milligrams). |
| Many labs that express proteins use prokaryotic cells | (such as E. coli); easiest to work with; take up plasmid DNA easily and divide very quickly; very easy to grow in culture and liters can be produced in a day at very low cost; high levels of protein expression (grams); |
| Some researchers choose eukaryotic systems for the expression of their recombinant proteins | Yeast and plant cells can produce large amounts of protein that are more likely to fold properly and have proper modifications; can be more difficult to work with as the DNA manipulation involved can be time-consuming, and the cells divide slower |
| Animal cells offer many advantages over other expression systems | allows for the expression of vary large proteins (up to 5000 amino acids, high yields, proper folding, post-translational modifications; disadvantage is that this system is not as straight forward; more expensive and longer wait involved |
| Mammalian cells can also be used for expressing recombinant proteins | can be used as a starting material for purification, and some researchers will obtain large amounts of animal organs and purify proteins directly from them; works well with animals, but is not feasible to obtain large amounts of human proteins |
| disadvantages to using mammalian cells | difficult to work with, yield are low, the process is expensive and time-consuming, there are special media and incubator requirements, and the systems have not been optimized in comparison to the prokaryotic system. |
| insect cells | High yield; Limitless protein size; Efficient cleavage of signal peptides, protein processing; Expression of multiple genes; More difficult than bacterial systems, may modify protein incorrectly or not secrete it |
| In the absence of lactose | the lac operon is turned off and the lac repressor protein is bound to the operator, which prevents E. coli RNA polymerase from transcribing the mRNA required to break down lactose. |
| the Ptac expression vector (such as the pGEX vector) | has a Ptac promoter that is silenced by the lac repressor protein; turned on by the addition of IPTG by removing the lac repressor; can be leaky that may result in misfolded proteins or death of cell |
| the phage expression vector (such as the pET vector), | has tighter control and wont have as much background expression; uses host proteins, bacteriophage proteins, and the lac operon;IPTG= on;T7 RNA polymerase targets the T7 promoter on the plasmid, transcribes the gene into mRNA, which is turned into protein |
| ammonium sulfate cut (salting out) | a controlled precipitation where proteins are gently removed from the buffer and stabilized by the positive charges of the ammonium and negative charges of the sulfate; used if chromatography was not successful or to clean up a lysate; cheap |
| disadvantages of ammonium sulfate cut (salting out) | can be difficult to obtain large amounts of pure proteins; may also aggregate with their natural binding partners; cannot be purified via affinity chromatography; difficult to study human proteins |
| Solvent precipitation | can be used to denature, inactivate, or remove unwanted proteins from a sample; useful for proteins that can withstand harsh chemicals but it usually saved for DNA and virus purification |
| Proteins are least soluble and often precipitate when | the pH of the solution equals their pI and may be used to selectively precipitate the protein of interest or to precipitate contaminants; most useful when pIs are different |
| inclusion bodies (aggregates)purification | accomplished with chaotropic salts such as urea and guanidine-HCl by decreasing the amount of tertiary structure in a protein and upon removal of chaotropic salts, the proteins will refold |
| Dialysis (or buffer exchange) | a process in which proteins are placed in special membranous tubing with very small holes that only allows the buffer and small molecules to pass through; each dialysis reduces concentration to 10% |
| Samples may turn yellow | as they approach high concentrations and at some point precipitate out of solution and can be countered (or even reversed) by increasing the salt concentration, which can help stabilize the protein, but only to a certain point. |
| Proteins can also be concentrated using lyophilization | they are dried under vacuum over several days to a fluffy powder; can be stored for years in the freezer |
| ultrafiltration | where proteins are placed onto a membrane that allows only buffer (sometimes small impurities too) to pass through; can be done using centrifuge tubes, or nitrogen pressure into a stirred cell. |
| Reverse dialysis | can also be used to concentrate proteins by placing the sample in dialysis tubing and surrounding it in polyethylene glycol to draw out the water and reducing the buffer volume. |
| exponential phase of bacterial growth | OD_600 = 0.6 |
| Size exclusion chromatography (SEC) | a gentle method for separating molecules based on size and can be used for desalting or buffer exchange; reproducible technique; disadvantage that the sample must be applied to the top of the column in less than 5% of the total column volume |
| Void volume | the proteins with larger molecular weights than the size of the beads that are eluded first from the SEC machine |
| Column volume | small proteins such as buffers and salt that must take the longest path through the column and therefore elute last. |
| Ion exchange chromatography (IEX) | a technique that separates proteins based on their charge; net charge of a protein is highly dependent upon the pH of the surrounding buffer. |
| isoelectric point (pI) | the pH at which there is an overall net neutral charge on the molecule that is calculated by the average of the pI of every one of its amino acids. |
| As the pH increases above the pI | hydrogens dissociate from the proton’s side chains and termini, leaving a net negative charge on the molecule and anion exchange can be used |
| As the pH decreases below the pI | hydrogens associate with the protein, giving it a net positive charge and cation exchange can be used |
| Anion exchange chromatography | involves exchanging negatively-charged anions accomplished by using beads with a positive charge; the buffer must have a pH higher than the pI of the protein to ensure it has a negative charge. |
| Cation exchange chromatography | involves exchanging positively-charged cations accomplished by using beads with a negative charge; the buffer must have a pH lower than the pI of the protein to ensure it has a positive charge. |
| Proteins will elute based upon (IEX) | the strength of their interaction with the beads – those that are in a buffer with a pH close to their pI will elute first, and proteins in buffers very far from their pI will elute last. |
| as the NaCl gradient increases, the elution diagram for anion exchange is | 1st peak: positively or neutral proteins; 2nd peak: low density of negative charge; 3rd peak: high density of negative charge |
| Hydrophobic interaction chromatography (HIC) | separates based on the lack of charge and is used when there are available hydrophobic amino acids on a protein’s surface; opposite of ion exchange so salt gradient is decreased to induce elution and buffer is highly polar; least hydrophobic elute first |
| Reverse-phase chromatography | (similar to HIC) is generally used on small molecules such as peptides, which are denatured and loaded onto a column containing long-chain hydrocarbons (C8-C18); Separation is based on their increasing order of hydrophobicity using methanol |
| Affinity chromatography | involves a molecule on a column binding specifically to a molecule in the sample; usually the first step in any purification, and so specific that it is often the only column necessary to purify a single protein from thousands in a lysate. |
| Affinity columns consist of | beads that contain immobilized small molecules or proteins that bind with high specificity to a single protein in a lysate by making use of antigen-antibody interactions |
| gene fusion technique | the gene for the protein of interest is fused with a linker to a second gene encoding a protein "tag" that is easily purified by affinity chromatography; not always preferable due to having to cleave the tag from the protein of interest with a protease |
| FLAG tag | is very small (DKDDDDK), and there are antibodies that specifically target it for affinity purification or for use in western blotting or cell staining |
| Metal-chelate chromatography (or IMAC) | is when nickel or cobalt (lower affinity, but it may be gentler and reduce contaminant binding) are loaded onto a column and proteins with histidines (his) tags are passed over the column; gene of interest is modified to encode a polyhistidine tag |
| Nickel-coated magnetic beads | can be used to purify his-tagged proteins directly in a microcentrifuge tube by mixing with his-tagged proteins to bind, and the beads are pulled to the side of the microcentrifuge tube with a magnet, washed, and the protein is eluted. |
| Intein-mediated affinity chromatography | a protein of interest is tagged to intein (an insect protein that binds to the glucose derivative chitin) via a disulfide bridge and washed over a chitin column. |
| Tandem affinity purification | is designed to contain multiple tags to improve purification |
| HaloTag | involves a tag on proteins that will form a covalent bond with a column that will provide the best purification as it can be washed indefinitely and eluted when one washes with a protease and his-tag that can be removed with a nickel column. |
| Gel electrophoresis | the separation of charged molecules through a gel matrix in order to sort them by size and/or charge at the buffer pH (If these two characteristics equalize migration may happen at the same rate) |
| Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) | allows proteins to separate on molecular weight alone and remove its charge as a factor in its migration on a gel; used to determine a protein’s molecular weight, identity, purity, and quantity, and is sensitive enough to distinguish ~1% apart in mass. |
| SDS (with the assistance of heat) | breaks weak interactions and causes the protein to unfold and linearize due to repulsion; |
| Sodium dodecyl sulfate (SDS) | Each SDS molecule has two negative charges and thus the net charge will be much more negative than normal; SDS binds to proteins in proportion to the number of amino acids; charge to mass ratio = 0.5 |
| mercaptoethanol (β-ME) or dithiothreitol (DTT) | break disulfide bridges |
| tracking dye (bromophenol blue) | a small, negatively-charged molecule that gives the protein samples a blue color; helps to see the samples when loading them into the wells of the gel; its movement also indicates that the current is actually moving through the gel and when to stop |
| glycerol | makes the samples more dense than the running buffer and ensures they sink into the wells; Without glycerol, the samples may disperse into the running buffer and be lost or contaminate other lanes |
| A loading buffer may contain | a loading control such as β-actin, which can be used for normalization to account for variations in pipetting between lanes after the gel is stained. |
| Acrylamide | is a small molecule that will polymerize into chains under the right conditions, and the porosity of the polymerized polyacrylamide gel can be controlled by introducing BIS-acrylamide |
| Ammonium persulfate (APS) | is necessary to catalyze the reaction between acrylamide and BIS and readily forms unstable SO4- radicals, |
| tetramethylethylenediamine (TEMED) | interacts with the APS radicals to induce the cross-linking and polymerization of acrylamide and BIS |
| Polyacrylamide pore size is determined by | the concentration of acrylamide; The higher the concentration of acrylamide in the gel, the smaller the pore size, and the more slowly molecule will move through the gel. |
| A gradient gel | can also be used in which the acrylamide concentration increases towards the bottom of the gel; This counteracts the logarithmic migration through the gel, and gradient gels can be used to separate proteins with a very wide range of molecular weights. |
| the stacking gel (~5% acrylamide) | has a lower percentage of acrylamide and is very porous which allows for the sample proteins to concentrate into a narrow band prior to reaching the resolving gel; Without it, the protein bands on a gel would be very wide and likely unreadable; pH 6.8 |
| resolving gel (~10-15% acrylamide) | has a higher percentage of acrylamide and actually performs the separation of the proteins; pH 8.8; only 10-40 micrograms of protein are added per lane |
| using tightly binding dyes to visualize SDS-PAGE | requires fixing proteins to the gel via ethanol to ensure they don’t migrate out of the gel, soaking the gel in Coomassie (which can be very messy) and destaining it with methanol; takes at least an hour or overnight; renders gel unusable downstream |
| using silver staining to visualize SDS-PAGE | 10-100X more sensitive than Coomassie, and offers nanogram detection; produces black or purple bands; difficult to perform by hand as it requires fixing the bands, staining, and multiple destains which can take hours; expensive and unusable downstream |
| using 2,2,2-trichloroethanol (TCE) to visualize SDS-PAGE | will react with tryptophan under ultraviolet light; allows for a very quick development (2 minutes) with microgram sensitivity; not as sensitive; no gel soaking required; can be used downstream |
| using autoradiography to visualize SDS-PAGE | This technique, while sensitive, is time-consuming and special precautions (and usually a certification course) must be taken when handling radioactive materials; isotopes also must be incorporated into the sample, which is not always possible. |
| To simplify molecular weight analysis | a standard curve can be determined using the markers (which have known molecular weights) on a gel with computer software; A protein band can then be selected and the computer will determine the molecular weight based on this standard curve. |
| a densitometer can be used | to measure the intensity of the bands, which would indicate a relative protein concentration compared to a known marker. |
| The relative mobility (Rf) of a protein can be used | for a more accurate molecular weight determination; is a ratio of the distance each protein migrates to that of the tracking dye (which has a high charge-to-mass ratio); negatively proportional to the log of the protein’s mass |
| Native gel electrophoresis | allows researchers to run proteins on a gel without denaturing them, therefore retaining their native structure and activity and will therefore migrate relative to both size and charge; pH 9; can't determine molecular weight; bands less defined |
| Native gel electrophoresis can be used to determine | a protein’s confirmation, quaternary structure (oligomeric state), analyze its structure/function, study protein-protein interactions, perform enzyme assays directly on the gel, and study protein modifications (phosphorylation, glycosylation, activation). |
| pore-limited gel electrophoresis | can estimate molecular weight with markers; resolving gradient gel of approximately 3-25% acrylamide is poured with no SDS; the proteins migrate until they reach a point where the pore size is too small and they can no longer proceed any further; |
| Isoelectric focusing (IEF) | allows the separation of proteins on charge alone; pH gradient is set up within the IEF gel by inclusion of various buffers and ampholytes; useful for determining the pI of proteins, and capable of very high resolution; expensive; proteins run until pH=pI |
| Two-dimension (2D) gel electrophoresis is a combination | IEF and SDS-PAGE; An IEF gel is run, and the resulting tube gel is then placed horizontally across the top of the stacking gel of a slab SDS-PAGE gel; proteins are separated by their pIs (IEF) and according to their molecular weight (SDS-PAGE) |
| Three-dimension (3D) gel electrophoresis is a combination | native gel electrophoresis, followed by IEF, followed by SDS-PAGE |
| Western blot (immunoblot) | a specific technique that allows one to detect and quantify proteins that react with a specific antibody; can be used to determine if a given antigen is present, and the concentration of the antigenic protein. |
| antibody | a protein that binds to a specific sequence of amino acids, or epitope; have specificity and affinity; produced from B-cells in the blood and lymphatic systems |
| western transfer | the SDS-PAGE gel is sandwiched between nitrocellulose (methanol activated) and blotting paper; soaked in buffer and the current is run perpendicular to the gel; proteins migrate out of gel and onto nitrocellulose in the same relative positions as SDS-PAGE |
| blocking step of western blot | ensures that all of the free protein-binding sites on the membrane are no longer available, and the antibody will only bind to the protein of interest. |
| Enzyme-linked immunosorbent assay (ELISA) | allows for the detection of an antigen or antibody in a sample; often used as a quick medical diagnostic tool; identical to western blot except an SDS-PAGE is not run beforehand; positive result=color change; can't determine molecular weight |
| indirect ELISA | has a setup identical to a western blot in that the antigen is bound to the membrane and a primary antibody is added to the well, followed by a secondary antibody; solution changes color when substrate is added to the well. |
| sandwich ELISA | uses an antibody-coated membrane that binds to the antigen; A secondary antigen coupled to an enzyme binds to the antigen at a second location; more specific and will reduce false positives; requires multiple antibodies that recognize the target antigen |
| Immunoprecipitation (IP) | involves precipitating an antigen out of solution using an antibody as a purification step |
| co-immunoprecipitation (Co-IP, or pull-down) | involves binding an antibody to a protein on solution, hoping to pull-down other proteins in complex with it to detect new protein-protein interactions |
| Specificity | ability to bind to specific target with low background |
| Affinity | how tightly the antibody binds to the target |
| Polyclonal antibodies | Multiple epitopes, lower specificity, higher affinity/signal, more tolerant of different fixation conditions; Produced by injection into animals (faster, cheaper) |
| Monoclonal antibodies | Single epitope, high specificity, lower affinity/signal, sensitive to fixation conditions for tissue specimens; Produced from hybridomas |
| Edman degradation involves | N-terminal sequencing of a very pure protein sample; bands can be cut directly from a gel or blot; limited to the first ~50-60 amino acids of a protein, but the first ~10 amino acids which should provide enough information for identification |
| Mass spectrometry sequencing | a protein is digested, and the molecular weights are analyzed and compared to known fragments; protein’s sequence can be determined by overlapping the fragments. |
| Protease digestion | another technique that allows for protein analysis, including domain analysis, conformation and activation analysis, and protein fingerprinting |
| far-western blot | allows one to identify protein-protein interactions; essentially a western blot with a few minor differences, such as a native gel and a radiolabeled bait protein probe; interactions can be detected by autoradiography |
| yeast two hybrid system (or bacterial two hybrid system) | a large library of potential partners can be screened at the same time; the system uses a report gene (lacZ) that is transcriptionally activated when the proteins interact and produces a color change in the yeast; |
| calorimetry | allows for the measurement of minute heat change in a sample that indicates binding or conformational change. |
| Isothermal titration calorimetry | measures heat change upon binding when one protein is titrated into a tube containing a binding partner; one requirement is that concentrations of the proteins that are interacting be known very accurately, often via amino acid analysis |
| differential scanning calorimetry | scans a protein across different temperatures and measures the results; can be very useful when comparing a protein vs. a mutant, as the two might behave differently at various temperatures. |
| surface plasmon resonance (SPR) | involves immobilizing protein on a membrane, running its binding partner across it to saturate it, and using light to measure this interaction; The rate of what associates vs. what dissociates is measured to determine the Kd |
| southwestern blot allows one to search for | proteins that bind DNA by using SDS-PAGE to separate the proteins, removal of the SDS, refolding of proteins with urea, and blotting the gel onto a nitrocellulose membrane; Next, digested labeled DNA is added, and DNA-binding proteins can be identified |
| Electrophoretic mobility shift assay (EMSA) | involves running DNA or RNA on a gel, followed by DNA with a potential binding partner and looking for a shift on the gel, which indicates a protein-DNA interaction; The protein can be identified by using an antibody and looking for further shifting |
| DNA footprinting | involves adding a protein to DNA and then digesting that DNA and running it on a gel to determine the exact sequence to which the protein binds; The protein will mask the sequence it binds, protecting it from digestion. |
| Chromatin Immunoprecipitation (ChIP) allows one to identify | the binding sites of transcription factors and other DNA-binding proteins; proteins are cross-linked to the DNA by formaldehyde, lysed, antibody-coated magnetic bead are added, a magnet is used to purify the antibody-protein-DNA complex |
| The general protocol for purifying DNA involves | disrupting cells, centrifuging the lysate to separate the various cellular fractions, adding proteinase K to degrade all of the proteins (including nucleases that can digest the DNA), and using various salts and organic solvents |
| EDTA | chelates metals and inhibits DNases |
| phenol-chloroform extraction | uses phenol (contains DNA and RNA), chloroform (contains proteins and debris), and isoamyl alcohol (to reduce foaming) to extract the DNA from the impurities; produces hazardous waste and any residual organic solvents will inhibit downstream steps |
| Alkaline lysis | uses SDS to break up phospholipid bilayer and NaOH to dissolve structure proteins. |
| Silica-based methods | allow the selective absorption of DNA to beads in the presence of chaotropic salts. |
| miniprep | based on the alkaline lysis and also involves a silica-based column that will selectively bind DNA; goal is to lyse and remove everything except plasmid DNA |
| An A260 of 1.0 = | 50 μg/mL of DNA in the sample. |
| A DNA sample with an A260/A280 ratio of | greater than 1.5 is considered to be relatively pure. |
| RNA is much less stable than DNA due to | its 2’ hydroxyl group that promotes degradation, and because of this RNases do not require metals and are therefore not inhibited by EDTA; RNA can withstand autoclaving; needs formaldehyde to run on an agarose gel |
| guanidinium isothiocynanate | denatures proteins and inactivates intracellular RNases |
| mRNA | has a polyA tail that can be used to purify via a polyT column; accounts for less than 5% of the RNA in the cell |
| agarose gel electrophoresis | very similar to SDS-PAGE; advantage is ease of preparation, DNA already has a built in negative charge so SDS is not needed; forms a thick stable gel; does not require a stacking gel; samples are loaded perp. to the current; can be a purification step |
| conformation of DNA and gel electrophoresis | supercoiled DNA will usually run more quickly than linear DNA (depending on the degree of supercoiling), and open circular DNA (or “nicked” DNA, plasmids with only a single ssDNA cut) will run the slowest. |
| pulse field gel electrophoresis | For very large DNA samples (>5000 bp); varies the current direction from multiple electrodes to increase sample separation. |
| UV shadowing | involves placing the gel directly on a UV; DNA, which absorbs at 260 nm, should appear as shadows in the gel; Unfortunately, a large amount of DNA is required to see any bands. |
| ethidium bromide | intercalates DNA, can be incorporated directly into the gel prior to polymerization, or gels can be soaked in it after electrophoresis; easiest and most reliable way to visualize DN; nanogram detection; carcinogen |
| SYBR green or methylene blue | Both bind to DNA like ethidium bromide, and are safer to use; These dyes do not offer the same intensity as EtBr when visualizing DNA and can only detect in the microgram range unless optimized. |
| autoradiography | radioisotopes like 32P are incorporated into the DNA and detected with X-ray film; This is a time consuming process and requires the use of radioactive molecules and special film developers |
| Recombinant DNA technology | term for combining DNA sequences that aren’t found together, which is very useful since it allows for the study of genes on a smaller, more controllable scale; process involves the biochemical manipulation of genes using enzymes |
| restriction enzymes | DNA-cutting enzymes; responsible for bacteriophage restriction; digest it at positions where adenines are methylated (Types I and III), and others at specific DNA sequences (Type II), which are more useful; used by bacteria as defense; named by source |
| Palindromic DNA | DNA that can bind to the complement of itself. |
| DNA methylases | naturally present in the cell and are used to determine the original vs. new strand during DNA replication; This methylation will inhibit DNA digestion by restriction enzymes, and protect the E. coli from its own enzymes |
| isoschizomers | restriction enzymes from different species that digest at the exact same sequences and in the same manner as other restriction enzymes from other species |
| neoschizomers | other restriction enzymes may recognize the same sequence of DNA, but cut in a different fashion |
| BSA | helps to stabilize and improve the efficiency of some enzymes as well as prevent the enzymes from sticking to pipettes and microcentrifuge tubes |
| Restriction enzyme tables | useful in determining if two enzymes have compatible buffers and can be used at the same time – if not the fragment must be digested with one, run on a gel, gel extracted, and digested with the other. |
| star activity | when a restriction enzyme is not always site-specific and can cut at an incorrect sequence; rare but increases if the reaction is allowed to proceed for too long or in an incorrect buffer |
| DNA polymerase | responsible for copying DNA during synthesis in the 5’→3’ direction, and making repairs upon DNA damage; can fill in sticky ends to produce blunt ends; requires magnesium |
| Klenow fragment | is often used instead of DNA polymerase I to fill in 5’ sticky ends, copying ssDNA, or preparing probes |
| TAQ polymerase | stable at high temperatures and used in PCR; very fast; lacks proofreading; adds A overhangs; makes mistakes |
| Pfu | outstanding proofreading capabilities; slow; rarely makes mistakes |
| for ligase to function | it must have two pieces of DNA that contain either blunt ends or overhangs that are complementary to one another. |
| sticky end vs. blunt end | Overhang “sticky” ligations are much more efficient than blunt end ligations because the base pairing of the overhands increases the chances of the two pieces of DNA finding each other and sticking together in solution. |
| Topoisomerase | can be used to ligate DNA; relieves tension during DNA replication by breaking the phosphodiester backbone of DNA, passing one strand across another, and reforming the bond. |
| cloning | placing a gene of interest into a plasmid, getting the plasmid into a bacterial host, and growing millions of clones of the original bacterial cell that took up the plasmid. |
| transformation | the process of taking up DNA from the environment; inefficient process, and very few cells actually take up DNA in an experiment, but only one cells needs to take up the vector as it can produce millions of copies of itself. |
| using CaCl2 to make competent cells | The calcium ions help to neutralize the DNA and the chlorine ions cause the bacterial cell to swell and create pores; The cells are mixed with the plasmid and heat shocked at 42°C for a short time, and chilled on ice; forces the plasmid into the cell. |
| Electroporation | shocks the cells with an electric current, causing the membrane to permeabilize and take up the plasmid; takes only seconds and gives a time constant that tells the researcher whether the transformation was likely successful or not; expensive, more prep |
| in vitro packaging | using bacteriophages to deliver DNA via transduction into E. coli |
| A replicon | a piece of DNA that is able to function like a chromosome in vivo and is capable of being replicated and partitioned stably over many generations. |
| Vectors (or plasmids or constructs) | dsDNA that are independent from the bacterial chromosome and have the ability to be passed from one generation to the next, and some can replicate in various host organisms; can be designed for cloning or protein expression; limited to under 50,000 bp |
| to be successful, vectors must have | an origin of replication (ori), a multiple cloning site (polylinker) that contains the gene of interest, a selectable marker, and some species require a partitioning sequence, such as the centromere in eukaryotes. |
| origin of replication (ori) | each chromosome must contain this in order be replicated and maintained stably in an organism |
| multiple cloning site (MCS) | polylinker designed to contain several unique sites for restriction enzymes located next to each other; |
| two enzymes with different cut sites is advantageous | because this prevents the plasmid from reannealing (and thus producing false positives), increases ligation efficiency due to the complementary overhangs base pairing, and ensures directionality of the insert during the ligation step. |
| blunt end digestions offer | versatility though, in that two different enzyme digests can be ligated together, and overhang digestions can be filled in or chewed back to blunt ends and ligated together; less efficient |
| selectable marker | ensures that the bacteria that grow are only those that have taken up and maintained the vector, and any non-transformants will die. |
| bacteriophages | can be packaged with the DNA of interest and mixed with E. coli. |
| Cosmids | combination of a plasmid and a lambda phage (and contain cos sites used for phage packaging), and are capable of replicating as a phage, in E. coli, and in eukaryotes; 50,000 bp and useful for making gene libraries |
| Bacterial artificial chromosomes and yeast artificial chromosomes | developed to carry hundreds of thousands or millions of base pairs of DNA. |
| Retroviruses | viral RNA is converted into DNA by the viral reverse transcriptase and is then integrated into the host genome, and any foreign or mutated host gene inserted into the retroviral genome will be integrated into the host chromosome as well; gene therapy |
| phenotypic screening | when a plasmid or cloned gene changes the phenotype of the cell in an obvious way; does not guarantee that the cells are also carrying the gene of interest; good initial screening |
| Conditional mutants | mutants that can grow under one set (permissive) of environmental conditions but cannot grow under different conditions (restrictive or nonpermisive). |
| Conditional lethal mutants | mutants that can grow under one set (permissive) of environmental conditions but die under different (restrictive or nonpermissive) conditions |
| Non-conditional mutants | display the mutant phenotype under all conditions, and require a supplement to survive; |
| A genomic library | contains all of the DNA of a cell in fragments |
| A cDNA library | contains the entire coding sequence and lacks introns and upstream regulatory regions |
| a standard cloning vector | designed for efficient uptake and replication in the host cell |
| a shuttle vector | can replicate in multiple hosts |
| an expression vector | allows for high levels of protein expression. |
| Probes | 32P- or fluorescently-labeled ssDNA that can hybridize (base pair) to a piece of ssDNA in a sample and tell you if that sequence is absent or present. |
| dot blot | The most basic application of a probe is to determine simply whether a given sequence is present or not in a sample of DNA; similar to ELISA; tells nothing of size |
| fluorescence in situ hybridization (FISH) | Probes can also be used to determine a gene’s locus on a chromosome |
| A Southern blot | a technique that not only tells you whether a piece of DNA is present or absent, but it also tells you the size of the DNA; similar to western blot; |
| Restriction fragment length polymorphism (RFLP) | allows one to identify whether an individual has a certain gene, or a different/mutant of the gene; needs a restriction enzyme specific to the polymorphic site; can be used to study recombination/crossing over |
| Variable Number of Tandem Repeats (VNTR) | relies on the fact that DNA can slip during replication or damage in repeat regions and this goes unnoticed by DNA polymerase and repair enzymes because the DNA still base pairs properly; replaced RFLP |
| northern blot | almost identical to a Southern blot, except instead of searching for DNA one is searching for RNA, and a labeled DNA or RNA probe is used. |
| RNase Protection Assay | a technique where a labeled probe is hybridized to mRNA creating dsRNA, and an RNase is used to chew up any remaining ssRNA; dsRNA is run on a gel and detected via autoradiography |
| DNA microarray results | red=a greater amount of mRNA, indicating that gene is up-regulated in normal cells; green=tumor mRNA is present in higher quantities, indicating the gene is up-regulated in tumor cells; yellow=both genes are equally expressed between the two samples |
| Polymerase Chain Reaction, or PCR | allows for exponential production of a piece of dsDNA |
| Watson strand= | strand on top |
| Crick strand= | strand on bottom |
| The forward primer | is identical to a section of the Watson strand and will bind to the Crick strand. |
| the reverse primer | is identical to a section of the Crick strand and will bind to the Watson strand. |
| primers | generally 20-30 nucleotides in length and should have greater than 50% GC content to improve bind to the template; bind to DNA between 100 and 1000 base pairs apart, facing one another. |
| melting temperature (Tm) | This is the temperature at which 50% of the primer is bound to the template DNA, and is determined by the number of AT and GC base pairs in the primer; can be estimated by adding 2°C for every AT pair and 4°C for every GC pair |
| annealing temperature | should be ~5°C lower than the lowest Tm of a pair of primers; too high causes the primer to not bind well and no copying; too low causes the primer to bind nonspecifically |
| quantitative PCR, or qPCR (also called real-time PCR or RT-PCR because samples are measured in real time) | measure the kinetics of the PCR reaction in its logarithmic phase; sensitive enough to detect 2-fold differences between samples; simultaneously quantify and amplify DNA |
| fluorescent resonance energy transfer (FRET) | that involves the transfer of energy between a donor and acceptor molecule; When the donor and acceptor are close in proximity, a transfer of energy occurs; when the two are far apart, no transfer occurs |
| SYBR green dye | fluoresces when bound to dsDNA, but not ssDNA; the advantage of being usable with any sample; disadvantage is that it will bind to any dsDNA, while probes are specific to a predetermined target and provide a permanent record of amplification; melt curve |
| melt curve | achieved by raising the temperature of the qPCR products slowly and measuring the change in fluorescence as the DNA dissociates; Multiple peaks indicate multiple samples of DNA; Peaks at lower temperatures indicate primer dimers or other contaminants |
| Ct threshold line | the point at which you (or likely computer software) choose to compare samples; can be used to compare samples to each other and determine the relative amount of starting material in the original samples |
| reverse transcriptase PCR, or RT-PCR (don’t confuse this with real time PCR) | uses reverse transcriptase, an RNA-dependent DNA polymerase, to synthesize DNA from an mRNA template; very sensitive and low amounts of starting mRNA needed; |
| reverse transcriptase quantitative PCR, or RT-qPCR (or RT RT-PCR) | the combination of qPCR and RT-PCR; replaced northern blotting for determining cellular mRNA |
| TA cloning | PCR products with A overhangs are directly ligated into a linearized vector containing T overhangs; avoids the need for a restriction enzyme digestion and gel extraction on your PCR products; does not guarantee insert directionality. |
| site-directed mutagenesis | uses traditional PCR and various primers to produce alterations in a gene product; requires two pairs of primers; |
| multiplex PCR | use more sets of primers and amplify multiple genes of interest; complex and time-consuming procedure that requires much optimization; |
| Touchdown PCR | involves starting PCR at a high, non-permissive annealing temperature and then incrementally lowering the annealing temperature in subsequent rounds; At some point, the primer with the highest specificity will bind and the target will be amplified |
| Nested PCR | increases the specificity of PCR by reducing nonspecific product amplification; uses four primers; primers 1 and 2 may have amplified a nonspecific product, primers 3 and 4 should only bind and amplify the gene of interest. |
| gene typing | allows a scientist to identify an organism’s genotype. |
| forensic DNA typing has replaced RFLP and VNTR analysis in paternity testing and crime scene analysis because | PCR is straightforward and requires very little starting sample; uses short tandem repeat polymorphisms that are highly variable to characterize various gene loci. |
| Amplified Fragment Length Polymorphism PCR (AFLP PCR) | circumvents the problem of not having an external primer to amplify a fragment of DNA by cutting DNA with restriction enzymes and ligating on adapter molecules; more sensitive, has higher reproducibility, can amplify up to 100 fragments at a time |
| Random Amplification of Polymorphic DNA (RAPD PCR) | uses short primers (~6 nucleotides) as priming sites for random DNA polymerase synthesis |
| ancient DNA | Modern techniques of DNA extraction permit the isolation of exceedingly tiny amounts of DNA; difficult to clone because of its very low quantity, contamination, and covalent modifications |
| asymmetric PCR | generates ssDNA probes via an asymmetrical primer ratio; useful for DNA hybridization and 32P-labeled nucleotides can be used |
| anchored PCR | allows for the amplification of gene when only part of the sequence is known and part is unknown or variable |
| Solid-state PCR | performs PCR in oil (emulsion PCR) or on beads (bridge PCR), from which DNA is easily retrieved; reduces cross-contamination, and does not require gel electrophoresis or cloning. |
| Helicase-dependent PCR | uses the enzyme helicase (as opposed to a thermocycler) to separate the two complementary DNA strands just as it occurs in the cell, and at lower temperatures than needed in traditional PCR; faster but requires more template DNA and optimization |
| Long PCR | PCR is limited to copying several thousands base pairs, and this can be overcome in some cases by using TAQ combined with Pfu and longer extension times |
| inverse PCR | can be used to amplify regions of DNA of unknown sequence that flank a known sequence |
| ligation-independent cloning | uses primer sequences that are complementary to a linearized plasmid |
| Maxam-Gilbert Sequencing | using a radiolabeled 5’ phosphate and 3’ cleavage of DNA with various chemicals; could be performed directly on DNA without the need for cloning or ssDNA; complex and time consuming; read bottom to top |
| Sanger Sequencing (or chain termination sequencing) | involves using dideoxy nucleotides (ddNTPS) that lack a 3’ hydroxyl group to terminate DNA synthesis; requires an acrylamide gel which can be read to obtain the DNA sequence (reverse and complement from bottom to top); 500-1000bp |
| Pyrosequencing (454 sequencing) | can sequence hundreds of megabases within hours by binding small fragments of DNA to beads immersed in oil and amplifying them via emulsion PCR. |
| Ilumina (Solexa) sequencing | uses bridge PCR to link primers to a solid support medium; As each base is added, it is detected with fluorescence |
| Ion semiconductor sequencing (or ion torrent sequencing) | works by adding dNTPs one at a time to the DNA to be sequenced; This changes the pH which is detected by a machine |
| Nanopore sequencing | does not directly measure fluorescence or change in pH involves passing a DNA sequence through a small nanopore and measuring the change in current; very fast |
| Microarray chips | contain millions of DNA oligonucleotides of various sequences; allows for very fast and efficient sequencing of large pieces of DNA, but is currently limited to sequencing only short fragments of DNA; if a spot lights up, the sequence is present |
| Mass spectrometry | used to sequence DNA since each nucleotide has a slightly different molecular weight; very fast but is currently limited to about 100 nucleotides |
| Cell culture | one of the most frequently used methods for cloning, gene expression, or studying cellular pathways; the media attempts to reproduce the cell’s native environment |
| E. coli model system | Inexpensive and efficient; however, they may not fold or be modified correctly |
| Yeast model system | It is also inexpensive and easy to work with, and can be used to study eukaryotic systems not found in bacteria, such as mitosis |
| baculovirus expression vector system (BEVS) model system | uses a virus to insert genes into an insect cell line; can express large proteins in high amounts that contain proper post-translational modifications; expensive and take 3-4 days to express the protein of interest. |
| mammalian model system | advantage being in their native environment, resulting in proper expression, folding; difficult to work with; require special CO2 incubators, the media must be changed every few days, and they are not as easily stored long-term; ethical issues |
| animal model system | most likely mimic the human form of a disease and response to a drug; But animals are expensive to house, require special care and FDA approval, and projects can take years to product useable data. |
| cell culture is an in vitro method | the incubation of biologically derived material in an artificial physical and chemical environment, outside of a living organism; disadvantage is that artifacts may appear |
| Artifacts | mechanisms or behaviors that only exist in a cell culture, and not in a living organism (ex: abnormally high H2O2 that may alter data). |
| Transfection | the process of inserting DNA into eukaryotic cells via via CaCl2 or other cationic polymers, electroporation, or heat shocking; is equivalent to transformation used on bacteria |
| genetically modifying T cell receptors | has been shown capable of targeting specific cell types to cure diseases such as HIV |
| RNA interference pathway | modify cells and silence protein expression; use siRNAs |
| Small interfering RNAs (siRNAs) | used to mediate sequence specific degradation of a target mRNA, thus shutting down translation; available to nearly every mRNA present in a human cell |
| Microinjection | technique that involves adding or removing nuclear material (enucleation) or injecting molecules into a cell via a small glass pipette being inserted into the membrane. |
| immunocytochemistry (ICC, cells) or immunohistochemistry (IHC), tissues). | antibodies recognize a specific protein while it is still in its native cellular environment; researchers can identify where proteins are found in the cell or tissue can track their expression and localization; similar to western blot |
| Pulse-Chase can be used to determine | metabolic activity by tracking a cell that is exposed to a radioactive molecule (Pulse), and after a few minutes exposed to a non-radioactive molecule (Chase); radioactivity can be tracked throughout the cell |
| patch clamp | allows scientists to suck up a small section of the plasma membrane in a pipette with an electrode inside that measures changes in current as ion channels in the cell open and close; recorded on an oscilloscope |
| Electrophysiology | the study of the electrical properties of a cell often by using microelectrodes |
| Cell sorting | the isolation in bulk of individual cells or populations of particular cells from a mixed population, which is often essential to research. |
| Flow cytometry allows a researcher to | separate cells based on size, health, or other properties such as cell stage or number of chromosomes; uses a vibrating nozzle to produce small droplets from a sample which then pass through a laser that detects various cell properties. |
| fluorescence-activated cell sorting (FACS) allows one to | sort cells based on light scattering and fluorescence; advantage is that it offers a researcher a wide range of properties upon which to sort cells;can be used diagnostically to determine cell ploidy or to determine a patient’s disease state. |
| vascular perfusion | technique involves fusing a compound (like formaldehyde and sucrose) through a fine hollow needle inserted into the artery carrying blood to the organ, and performing subsequent analysis on the blood being transported |
| Whole body autoradiography (WBA) | can be used to trace the localization of the drug in the frozen animal slice to determine if it binds to the tumor or not. |
| C. elegans (roundworm) | has the same basic organs and body plan as all animals in only 959 (hermaphrodite) or 1031 (male) cells; transparent; easy to disrupt gene expression via RNA interference |
| Drosophila (fruit fly) | very useful to researchers studying genetics |
| Zebrafish | very useful to researchers studying development |
| disadvantage to using animal studies | they don’t always produce human-like patterns of pathology, or respond in the same way to a particular treatment |
| Transgenic animals | have manipulated genomes or carry genes from another species; |
| Cloning | the process of creating an exact copy of a living organism; extremely inefficient, and it often takes hundreds of attempts for a single clone to take hold |
| knockout mouse | involves deleting a gene and is extremely useful in determining the gene’s function in the cell |
| knockin mouse | involves inserting a gene and is extremely useful in determining the gene's function in the cell |
| knockdown mouse | partial gene disruption, which may be needed if a gene is embryonic lethal if missing |
| Cre-LoxP mouse (or conditional knockout) | involves knocking out a target gene only in a specific cell type; Cre mouse contains Cre recombinase, crossed with a LoxP mouse, which has the gene of interest flanked by LoxP sites;Cre recombinase and will seek out the LoxP sites and remove the gene |
| optogenetics | When light strikes the neuron, channelrhodopsin opens and the neuron fires; allowed researchers to control a mouse’s movements with light; potential to one day be used for two-way communication between the brain and prosthetics. |
| chromosome walking | genomic DNA carried in a clone is sequenced, and a fragment of this sequence is used as a probe to isolate the next clone, which is sequenced next; sound in theory, but at the time only 500-1000 bp of DNA could be accurately sequenced at a time |
| shotgun sequencing | the human genome is mechanically sheared, polished to add adenines to each piece of DNA, incorporated into plasmids via TA cloning, and sequenced using plasmid-specific primers; the sequenced fragments were then pieced back together on a computer |
| National Center for Biotechnology Information | can search research publications, nucleotide and protein sequence databases, Mendelian genetics databases, protein structure databases, and more. |
| GenBank | a database that contains DNA sequences amounting to billions of base pairs and DNA from thousands of different organisms, from viruses to humans |
| PubMed | a searchable database of thousands of scientific journals; first place one should visit when looking for peer-reviewed scientific data on a topic or when performing a full literature review for a research project |
| the Basic Local Alignment Search Tool (BLAST) | an excellent tool for comparing any sequence with which you are working to the entire GenBank database; useful to find similar DNA or protein sequences among various species; if two sequences are ~30% identical or more it indicates similar function |
| Expert Protein Analysis System, (ExPASy) | contains a collection of tools and software used to analyze DNA and protein sequences |
| Protein Data Bank (PDB) | currently contains over 70,000 protein structures, with dozens added daily |
| Chou and Fasman | Their analysis of proteins showed that certain amino acids were more likely to be involved in α-helices, β-sheets, and turns than others; 50% accurate |
| Garnier-Osguthorpe-Robson (GOR) | analyzes a larger segment of amino acids (17 amino acids total) to predict secondary structures; 70% accurate |
| Ramachandran Plot | analyzes the phi and psi angles around the α-carbon of an amino acid; Certain combinations of phi and psi tend to be found in specific secondary structures; useful for predicting secondary structures, but also used as a refinement tool |
| α-helices | generally have a phi and psi near -60 and -50, respectively |
| β-sheets | generally have a positive psi |
| helical wheel | Protein sequences can be analyzed to look for this pattern of alternating residues to not only discover new α-helices, but predict their location and possible function in the overall protein sequence; full α-helical turn is approximately 7 amino acids |
| hydrophobicity plot | assigns a number to each amino acid based on how hydrophobic or hydrophilic it is; useful in predicting transmembrane proteins |
| disorder plot | looks for regions of high movement or disorder |
| molecular mechanics | technique uses the atomic nuclei and force fields to predict structures and interactions; not a perfect representation of what is occurring at the molecular level. |
| stimulated annealing | technique essentially decreases the temperature of the molecule in the computer; the molecule will vibrate greatly, and as it “cools” it will hopefully find the energy minimum, or the most favorable (and most likely) structure. |
| molecular dynamics | allows for one to model molecular interactions, like a protein with a binding partner, myoglobin with oxygen, an enzyme with a drug, or modeling all proteins in the cytoplasm |
| virtual screening | an efficient way to screen thousands of potential protein inhibitors on a computer; cost-effective, reduces false positives, increases the number and diversity of potential inhibitors, and helps to find new classes of drugs |
| proteomics | the study of all of the expressed proteins in an organism, including various isoforms and modifications |
| genomics | is the study of an organism’s entire genome, which involves genome sequencing, genetic mapping, studying gene regulation and expression, and evolution and gene conservation. |
| mass spectrometry | allows one to determine the mass of a molecule with extreme accuracy – to the thousandths place and greater; most involve an ionizer, a mass analyzer, an ion detector, and a mass spectrum |
| Electrospray ionization | where a protein sample is sprayed with a charged solvent and detected. |
| Liquid chromatography mass spectrometry (LC-MS) | injects a liquid sample of proteins directly into the mass analyzer |
| Matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) | uses a laser to greatly ionize large molecules |
| Fluorescence microscopy | allows researchers to view aspects of the cell not visible with a normal microscope; proteins can be tracked as they move throughout the cell when they are tagged or encoded with fluorescent dyes/chemicals |
| Confocal microscopy | similar to fluorescence microscopy but uses lasers to greatly improve the image quality and resolution |
| Transmission electron microscopy (TEM) | the electron beam goes through the sample; allows for high magnification |
| Scanning electron microscopy (SEM) | the electron beam bounces off of a gold-plated sample; more useful for 3D images as it only reveals the surface of the sample. |
| cryo-electron microscopy, or cryo-EM | copies of a sample (like a protein or virus) are frozen and placed under an electron microscope; Hundreds or thousands of pictures are taken from various angles, and these images are reassembled into a single image of up to 4-angstrom resolution |
| Atomic force microscopy | technique developed to visualize small molecules and proteins; device is similar to a tuning fork that vibrates near molecules, causing a vibration that can be measured. |
| Circular dichroism (CD) | a technique that allows one to get an idea of the secondary structure feature of a protein; measures differences in the absorption of the left-handed polarized light versus right-handed polarized light; secondary structure can be inferred |
| nuclear magnetic resonance (NMR) | technique uses a strong magnetic field to determine the relative positions of certain atoms in the protein; study molecules directly in solution without the need for a crystal; limited to proteins under ~30 kDa in size |
| X-ray crystallography | can be performed on anything from a small molecule to very large proteins, so long as a crystal of the molecule is available |
| asymmetric unit | the most basic unit of a crystal, which for example may be a single molecule or protein |
| space group | the orientation of the crystal’s subunits, and they are broken down into 7 major groups based on symmetry |
| Fourier transform | converts imaginary space to real space; the data from the hundreds of images of the crystal can be integrated into a single dataset and its structure can be determined. |
| Each spot on a diffraction pattern of a crystal | represents X-rays that interacted with electrons on the proteins and were diffracted via constructive interference (in phase); this data is used to generate an electron density map |
| phase problem | scientist must determine the phase at which the light wave struck the crystal to solve the structure; concern because the majority of “image” information is carried in the phase, not the intensity (which is detected by diffraction) |
| phase problem solution | molecular replacement, a known structure is used to solve an unknown structure; isomorphous replacement where heavy metals are soaked into the crystal and their diffraction is located in the data; |
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