Question

Please Help with simple explanation ..

3654 Biochemistry Expt. 7 (cont.) Name with reducing agents A B A B (5pts) Protein A contains 2 polypeptide chains (1 of 70,000, 1 of 35,000) held together by disulfide bonds. Protein B contains 2 polypeptide chains (each -75,000) held together by hydrophobic interactions and electrostatic forces. On the SDS-PAGE gel to the right, sketch the ideal pattern you expect for each of these proteins in the absence and presence of reducing agents. (Use your text for help if you need it.) Explain briefly your logic. 150,000 100,000 65,000 50,000 35,000 25,000 (2pts) What does the protein trypsin do to other proteins? Sample Sample 2 (3pts) In the space to the right, draw what you expect for the gel electrophoresis pattern of acid phosphatase if one sample was treated as in this lab (sample 1) and another acid phosphatase sample was incubated briefly with a small amount of trypsin (sample 2) prior to SDS-PAGE. Make sure your picture is clear and thorough. Acid phosphatase has a molecular weight of approximately 58,000 Daltons (g/mol). Briefly explain why you drew your picture the way you did. (4pts) Explain why lactoglobulin traveled further than BSA during SDS-PAGE but traveled about the same distance during agarose gel electrophoresis.

please answer at least the last 3 questions if can't estimate ,, please answer the first Q only

Answer 1

with our computent with a reducing LA B A B 1 150.000 + 100.000+ 65,000 50.000 35,000 25000

Protine A contains two polypeptide chains (70000+35000) held together by disulfide bond.

Protein B contains two polypeptide chains ( 75000+75000) held together by hydrophobic interaction and electrostatic force.

SDS PAGE is a type of gel electrophoresis. By this process larger molecules are separated by their molecular mass and charge. In SDS PAGE, sodium directly sulfate is a detergent. It can break hydrophobic interactions, hydrogen bonds but cannot break disulfide bonds.

Reducing agents can break disulfide bonds.

So when reducing agent is absent Protein A remains intact and protein B breaks into two chain of same weight . That is why protein B gives only 1 band.

When reducing agent is present, protein A splits into two polypeptide chains of different molecular weight. So they gave two different bands.

Trypsin is an proteolytic enzyme mostly found in vertebrates. It produced by pancreas and it breaks protein molecules at specific site. Trypsin mainly breaks peptides in carboxyl end of amino acid lysine or arginine. It mainly helps in protein digestion.

Lactoglobulin has a molecular weight of 18.4 kDa. It is a small protein .

BSA has molecular weight of 66.5 kDa.

SDS PAGE can separate proteins based on their mass so and charge. So, lactoglobulin traveled further than BSA.

But agarose gel electrophoresis separates proteins based on size and charge. Agarose gel can separate high molecular weight proteins perfectly. So in case of agarose gel electrophoresis both proteins traveled same distance.