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EB 3410 Chapter 7
EB3410 Manipulating Biomolecules: Proteins Western Blotting :Quiz 2
| Question | Answer |
|---|---|
| Is it just genes that determine what proteins will be made? | Current research in the field of proteomics suggests not |
| Proteins are essential to maintain the ____ and ____ of life forms | Structure; function |
| _______ is the study of proteins, particularly their structers and funcitons | Proteomics |
| While the genome is a rather constant entity, the proteome differs from _______________ and is constantly changing through its biochemical interactions with the ___________ and ________ | cell to cell; genome; the environment |
| Each and every property that characterizes a living organism is affected by _____ | Proteins |
| T or F: the absence of one or more proteins in the system can cause life-threatening diseases | True |
| How many amino acids are there? | 20 |
| Protein function is determined by the diversity of the ________ as well as the ______ that the building blocks can form | 20 amino acids; 3D structure |
| What is the overall charge of a protein after it is prepared for loading on an SDS PAGE gel? | negative |
| What are the 4 fundamental non-covalent interactions? | (1)Ionic Bonds (2)Hydrogen Bonds (3)Van der Waals forces (4)Hydrophobic Interactions |
| Why is it important to know the concentration of protein in a sample? | (1)Obtaining reliable, repetitive results (2)Determining protein purity and activity |
| How is Protein Concentration applied? | (1)Characterization and purification of proteins and enzymes (2)Physiological studies of protein expression (3)Clinical diagnosis of altered protein levels in bodily fluids (indicates diseases) |
| T or F: There is one method of protein determination that works for all applications | False; no single protein determination methods is suitable for ALL applications |
| What are some factors to consider when choosing a [protein] assay? | the range of the useful response; sensitivity; specificity; ease; safety considerations; cost |
| What are the 5 methods for quantification of proteins? | (1)A280 (2)Biuret (3)Lowry (4)BCA (5)Bradford |
| What piece of equipment is needed for all 5 methods of protein quantification? | A Spectrophotometer |
| Which protein assays are used more frequently? | A280; Lowry |
| Which protein assays are highly sensititve? | Lowry; BCA; Bradford |
| Which protein assays are inexpensive? | A280; Lowry |
| Which protein assays are compatible with most buffers? | Bradford |
| What is a con of highly sensitive assays? | They are also sensitive to contaminants |
| Which protein assays yield a standard curve of non linear protein concentrations? | Bradford |
| Which protein assays require a cure to be run with each sample and can stain cuvettes? | Bradford |
| Which protein assays have highly reproducible results? | Lowry |
| Which protein assays are very rapid? | A280 |
| Which protein assays do not depend on the amino acid sequence? | Biuret |
| Which protein assays are not very sensitive? | Biuret |
| What is the difference between Native PAGE and SDS PAGE? | Native=proteins are run in native conformation; SDS PAGE=linearized protein |
| What interactions need to be broken in order to linearize a protein? | 4 non-covalent interactions (hydrophobic interactions; van der waals; h-bonds; ionic bonds) and disulfide bonds |
| Which reagents are used to break disulfide bonds? | DTT or BME |
| What will give the unfolded protein the overall universal charge (what is the overall charge) | The binding of SDS; negative charge |
| T or F: During Native PAGE electrophoresis, proteins with the same molecular weight will migrate differently through the gel | True |
| What determines the migration distance of proteins in Native PAGE? | mass to charge ratios |
| T or F: During SDS PAGE electrophoresis, proteins with the same molecular weight will migrate differently through the gel | False, they will migrate similarly in an electric field |
| Both the ______ method of the support medium and the _____ of the molecules contribute to the separation process. | sieving; mass/charge ratio |
| ________ permits the separation of molecules of identical charge/mass ratios, but of different sizes | SDS-PAGE |
| Why is the protein sample heated in the Laemmli buffer? | Promote denaturation -- Liniearize; ensure SDS & BME/DTT contacts every region |
| What does the loading buffer contain? | Glycerol, bromophenyl blue, and SDS |
| What purpose does glycerol serve in the loading buffer for an SDS-PAGE gel? | weighs down sample |
| What purpose does bromophenyl blue serve in the loading buffer for an SDS-PAGE gel? | indicator of migration distance in the gel |
| How is pore size changed in an SDS-PAGE gel? | Altering the ratio of acrylamide to bisacrylamide (a higher [] crosslinker will yield narrower pores) |
| What reagents are used to prepare the polyacrylamide gel? | (1)Acrylamide-momomer (2)Bisacrylamide-crosslinker (3)APS (4)TEMED (5)Water/Buffer |
| What purposes to APS and TEMED serve in making the gel? | Create free radicals - link monomer and crosslinker |
| Are there any safety hazards with the gel reagents? | Yes. Acrylamide is a nuerotoxin in its unpolymerized state |
| Why is cross-polymerization important when preparing a polyacrylamide gel? | Without it, the solution would be viscous, and would not form a gel (long chains sliding over each other) |
| At what stage in the preparation of the polyacrylamide gel should APS and TEMED be added? | Right before pouring the gel, because the rate of polymerization is high (fast) |
| Usually _____ to ______ of a protein mixture is loaded onto a gel. | 15ug-20ug |
| What is required for obtaining a sharp, focused band on an SDS-PAGE gel? | a stacking gel casted on top of the resolving gel |
| What is a low-percentage gel prepared with a buffer with a pH of 2 units lower than electrophoresis buffer | Stacking gel |
| T or F: Stacking gels allow concentration of protein-SDS complexes to a thin starting zone in a short period of time | True |
| Stacking gels permit effective and reproducible separation of the SDS-coated coated protein by _____ | size |
| Stacking gel has a _____ % of acrylamide to insure ____ porosity and a resolving gel has a _____ % of acrylamide to insure _____ porosity | low; high; high; low |
| T or F: concentration of protein to a thin starting zone takes a long time | False: a short period of time |
| T or F: The resolving gel is made with a Tris-HCl buffer at a pH of 6.8 and the stacking gel is made with a Tris-HCl buffer at a pH of 8.8 | False: Resolving gel = pH 8.8 and Stacking gel = pH 6.8 |
| 1aa = ____Da | 110 |
| T or F: Proteins migrate differently on different percentages of poyacrylamide | True |
| What are gradient gels? | Polyacrylamide gels that vary in percentage throughout; transition from low to high (do NOT have a stacking gel) |
| The upper and lower reservoir buffers usually contain which buffer? | Tris-glycine-SDS |
| What buffer is used to make the acrylamide gel? | Tris-HCl |
| T or F: Since the stacking gel is very pourus, there is little difference in the mobility of different size proteins as they migrate through it | True |
| At what temperature is the protein sample stored before used in the SDS-PAGE gel? | -20 degrees C |
| What volumes are loaded into the wells of the gel?--- to try to achieve a band that can be used for antibody detection | 1: 10uL, 2: 15uL, 3: 20uL, 4: 25uL |
| What is the first step in casting a gel? | Making sure glass plates are clean and dry (spray with 70% EtOH) |
| Dry the top of the resolving gel with __________ before pouring the stacking gel. | filter paper |
| How long should a stacking gel polymerize for? | 30-45min |
| Why is a buffer needed in the gel chamber? | Ions need to be present to conduct electricity |
| on average, there is ___ SDS molecule(s) for every ____ amino acid(s) | 1 SDS: 2aa |
| (In SDS-PAGE) the separation of the denatured, detergent-coated prolypeptide chains will be due almost exclusively to the _____ of the polypeptide chains | Size |
| The _______ will be essentially identical for different proteins because the SDS coating dominated the ___ | charge/mass ratio; charge |
| T or F: proteins hae a net charge at any pH other than their isoelectric point | True! |
| Why is a support medium used? (gel running) | to prevent the loss of resolution due to diffusion, vibrational and convectional disturbances, and to retain the separation of different proteins after completion of the experiment |
| What are some properties of a support medium? | Strong; hydrophilic; uncharged or stable over Temp., pH, or osmolarity changes; carefully controlled porosity |
| What are 3 examples of stains to detect the presence of proteins? | (1)Coomassie Brilliant Blue R-250 (2)Silver Stain (3)Radioactive Labeling |
| rank the protein stains from least sensitive to most sensititve | Coomassie -- Silver Stain --- Radioactive labelling |
| About how long does a coomassie stain need to resolve? | 1 hour |
| About how many times more sensitive is silver over Coomassie Brilliant Blue | 10-100x |
| T or F: Silver requires a shorter staining time than Coomassie Brilliant Blue? | False; longer time (but the longer the better resolution) |
| What is a disavantage of radioactive labelling? | Not clear bands |
| What is the range of the molecular weight of proteins? | 10-300 kDa |
| Mobility of the protein is a ______ function of the logarithm of of its molecular weight. | linear |
| What are some applications of SDS-PAGE? (x4) | (1)Routine analysis of the purity of protein samples (2)Determination of the molecular mass of polypeptide chains (3)Immunoblotting (4)Purification of denatured proteins |
| What are some more applications of SDS-PAGE? (x4) | (5)Native gel electrophoresis (6)Gradient Gel Electrophoresis (7)Isoelectric Focusing (8)2D gels |
| What is used to detect and quantify proteins that react with a specific antibody? | Western Blot! (Immunoblot) |
| T or F: Western Blotting is so sensitive that it can be used to detect the amounts of proteins not possible with other techniques | True |
| What are some characteristic of the nitrocellulose membrane used in the antibody sandwich? | Postively charged; can sustain multiple washes and incubation steps; provides a white background for colorimetric detection of the protein |
| In Western Blotting, what is the importance of the Blotting Paper/Filter Paper? | Support the gel and nitrocellulose/ protect them from the fiber pads during assembly; helps uniform flow of buffer |
| In Western Blotting, what is the importance of the Fiber Pads/Supporting Pads? | Press gel and nitrocellulose together/eliminate air bubbles; allow efficient transfer of proteins from the gel to the blot |
| T or F: SDS partially denatures proteins | True |
| SDS causes the protein to gain a negative charge ____________ to the protein's chain length | proportional |
| Why are colorimetric methods most commonly used for protein determination? | (1)widespread availability (2)great accuracy (3)relative specificity (4)general low cost, simplicity, and safety |
| Which protein determination method won't work for proteins that lack tryptophan, tyrosine, and phenylalanine | A280 |
| What happens to a protein when it is placed in a pH higher than its pI? | protein has an overall negative charge |
| What happens to a protein when it is placed in a pH lower than its pI? | protein gains an overall positive charge |
| What happens to a protein when it is placed at a pH equal to its pI? | the protein is overall neutral |
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