me: - Section: Date olyacrylamide Gel Electrophoresis In whiff typeof polyacryla
ID: 150095 • Letter: M
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me: - Section: Date olyacrylamide Gel Electrophoresis In whiff typeof polyacrylamide gel system do you separate denatured burt for se pevr reselv enatare Sth is on top resolving gel-s at the betYo, 8. What is the difference between the information obtained in a Coomasie-stained polyacrylamide gel and a Western blot? Why? acrulamicd Coomasie Sta but n western blot ue can ire proten bylecday wnight its rmole use eoit,pe which is anflmely regagnition site e ll u·tor certain, Proten d interest out Coomasie canno te Wester olot can. 9. A Coomasie stained gel cannot tell you for certain if your protein a. what do you need to do to be certain your protein is present and of interest is present. Why? Coomasie stainee ge est stains ta retiin bue. cesn t cer taim or nde elar wea1 nterest, ht , sa rt Can't tellor certain protein 07 inferest rotein gou shd use western blot 1o. Why is unpolymerized acrylamide hazardous? 2- hazardous because it handle it crfy s neure toxin , So f is ueaY ovesExplanation / Answer
7. SDS-polyacrylamide gel electrophoresis separates denatured protein exclusively based on molecular weight. Sodium dodecyl sulphate or SDS is an anionic detergent that breaks down disulphide linkages that result in loss of secondary and tertiary structure. This result in denaturation. SDS also binds to the linear protein and confirms it a net negative charge. This negative charge masks the R groups in proteins . Around 1.4 g SDS binds to 1g of protein.
In native or non-denaturation gel electrophoresis, there is no SDS added. Hence, no denaturation occurs and the proteins are in their folded structures.
Right answer is SDS- polyacrylamide gel electrophoresis or SDS-PAGE.
8. In Coomassie blue staining, the dye Coomassie blue binds to proteins via ionic interactions. These ionic interactions occur through Vanderwaal interaction between the sulphonic groups of the dye and the positively charged amino groups on proteins. Thus, Coomassie blue will bind to all proteins present in the mixture. The proteins are visualized as blue bands on the separated SDS-PAGE gel.
Western blotting involves transfer of proteins that are separated by SDS-PAGE on to a nitrocellulose or PVDF membrane. These membranes/blots are then treated with an antibody against the epitope of protein that is to be detected. Epitopes are regions on the protein that bind to the antibody. A secondary antibody against the primary antibody that is linked to an enzyme like horse raddish peroxidase is then added. Detection is through conversion of 3-diamino benzidine and hydrogen peroxide to brown coloured compound. Hence, only the protein of interest is detected.
Coomassie blue staining shows multiple bands while western blotting reveals the specific band of protein of interest.
9. Coomassie blue staining does not involve the use of specific antibody against the epitope of the protein that needs to be detected. It will bind to amino groups of all amino acids in the protein. Hence, all proteins in the sample are stained by Coomassie blue. As no specific antibody is used, the protein of interest cannot be detected.
The specific protein needs to be detected as it will indicate whether there are any changes in amount of the protein in the cell. Based on the amount of protein preset, it can be said that the protein is involved in pathogenesis of the disease. Western blot will identify the disease in the sample as it involves use of the antibody specific to protein. It will also indicate accurate concentration of the protein in the mixture of other proteins in the sample/cell. Housekeeping genes are simultaneously also estimated to provide quantitative measure of the specific protein amount. Decreased or increased concentrations are correlated with pathogenesis of diseases. Further, western blot will indicate the molecular weight of the protein.
10. Acrylamide is an odorless crystalline solid that is a neurotoxin. It can be absorbed through the skin. Acrylamide directly inhibits presynaptic transmission of neurons. It binds to the cysteine sites on proteins. Hence, it should be handled with care using gloves on hands.
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