Question

chosen for the first two steps of the purification while the Bradford assay was E) Why was the Biuret assay chosen for the first two ste chosen for the second two steps of the purification? (2 points) A the last 2 steps are coepon which is eleited 9 Bradford assay binds to su peptide bonds so the we can get zu accurate result you pour both native PAGE and SDS-PAGE yels. Besides chemicals must be added with water and buffer to create F) Finally you think your enzyme is pure, so you pour both native PAGE and Sps acrylamide (and SDS), what three other chemicals must be added with w polymerized gels, and what is the purpose of each chemical? (6 points) G) You electrophorese your purified sample on both the native and SDS-PAGE gels. Give an explanan for the difference in banding patterns seen on these two gels. (3 points) Native PAGE SDS-PAGE

Answer 1

(E) 1. In protein purification, for the first two-step, biuret assay was chosen because of to assay protein samples of unknown concentration and to pipet accurately.

2. Bradford assay can be used to determine the concentration of proteins in solution. The assay is based on the formation of a complex between the dye, Brilliant Blue G, and proteins in solution.

(F) 1. N, N'-Methylenebisacrylamide is a cross-linking agent

2.  Tetramethylethylenediamine is a catalyst.

3. Ammonium persulfate is a radical initiator.

These three chemicals must be added in water and buffer solution to start the polymerization, further, this solution is poured between the glass plate without forming bubbles.

(G) 1. In Native Page, separation is based upon the charge, size, and shape of a sample while SDS separation is based upon the molecular weight of the protein sample.

2. The native page is best for separation or purification of protein from a mixture of protein and the SDS method is used to determine the weight of proteins.

3. As we have seen in both of electrophoresis, we poured two samples in wells, one side we got 5 bands Same in both the electrophoresis mean the first sample is the same but sample poured in second well, we got only one band but different size or molecular band this analysis suggest that the first sample is same in both but the second sample is different.