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BSC 3403
Exam 2
| Term | Definition |
|---|---|
| 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. |
| end result of such DNA manipulations | the insertion of a human gene of interest into a bacterial plasmid |
| plasmid (also called vector) | called recombinant DNA since it contains DNA from various species and is not natural; can then be transformed into bacteria where the gene can be expressed. |
| 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 |
| 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 |
| efficiency of lysing cells (most to least) | French press, sonicator, chemical lysis, lysozyme, freezing/thawing, mortar and pestle or blender |
| 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 |
| recombinant DNA technology | Advantages: Can obtain exact protein in large amounts; Can engineer mutants; Cost-effective Disadvantages: Must know DNA/protein sequence; Cloning/purification can be tricky; Proper folding?; No post-translational modification |
| Partition chromatography | a process that uses multiple liquid solvents to separate material. |
| Chromatography | involves the separation of a substance by using a stationary phase (such as beads) and mobile phase (sample and buffer). |
| Gas chromatography | study small molecules by passing them in a gaseous state over a stationary solid absorbent and the time it takes for a molecule to elute from the column is measured. |
| Liquid chromatography | separate mixtures of molecules in liquids (such as metals or organic molecules) form by passing them over a column |
| Thin layer chromatography | allows one to separate, analyze, and check the purity of organic compounds such as fatty acids, pesticides, and drugs |
| High performance liquid chromatography (HPLC) | developed in the 1970s, uses high pressure pumps to separate, identify, and analyze the compounds that are present in a sample with outstanding resolution |
| 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 |
| Stokes radius | the effective radius a molecule has as it tumbles rapidly in solution (i.e. the radius of a hard sphere will have a smaller stokes radius than a rod-shaped molecule) |
| 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 |
| Diethylaminoethyl (DEAE) and quaternary ammonium (Q) | are positively charged beads and are used as anion exchangers. |
| Carboxymethyl (CM) and sulfonic acid (S) | are negative charged beads and are used as cation exchangers. |
| 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 |
| 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. |
| Electrophoresis | the movement of charged molecules toward an electrode of the opposite charge – anions move toward the anode (+ electrode) and cations move toward the cathode (- electrode). |
| 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. |
| If proteins of low molecular weight are being analyzed | a gel of a high percentage of acrylamide is used; only 10-40 micrograms of protein are added per lane |
| If proteins of high molecular weight are being analyzed | a gel of low percentage of acrylamide is used; only 10-40 micrograms of protein are added per lane |
| 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 |
| 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 |
| Glycosylation | modification where sugars are attached to proteins, which can account for up to ~50% of a protein's mass |
| 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 |
| If the sequence is unknown | a researcher can enter it into a BLAST search, which will compare it to all known protein sequences of various species. |
| 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 |
| protease | an enzyme that cleaves a protein at a specific amino acid sequence; can also be used to analyze protein interactions |
| A full length protein may be resistant to proteases, but the addition of binding partners | can cause the protein to be more susceptible to protease cleavage, indicating that the binding partners lead to protein conformational changes and activation |
| 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 |
Created by:
JacobGant
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