3) You are studying a protein that you think consists of a heterodimer composed

Question

3) You are studying a protein that you think consists of a heterodimer composed of a 30 kDa and a 40 kDa subunit, which are joined together by disulphide bonds. In addition to your protein sample you have available a SDS-PAGE gel, an electrophesis apparatus like the one you used in the laboratory, and a Bio-Rad Precision Protein Kaleidoscope standard (Bio-Rad catalogue number 161-0375). You also have access to two different sample buffers. Buffer A contains 4% (v/v) SDS and 5% (v/v) ß-mercapto ethanol. Buffer B contains 4% (v/v) SDS but no ß-mercapto ethanol. As per the practical, samples are heated at 95oC for 4 minues before they are loaded onto an SDS-PAGE gel and electrophoresed.

Describe the experimental methods that would allow you to test your hypothesis. Provide sufficient detail such that an interested reader could faithfully repeat your experiment. In your answer describe the chemistry and biochemistry involved in your treatment.

(You determine hypothesis)

Explanation / Answer

Hypothesis Protein linked by di sulphide bond. to check this Hypothesis we divide protein sample into two parts. 1. one part of protein soln added to Buffer soln. A. While other added to soln B.

Heat the samples at 95 c for 4 min.

The gel soln is poured betn two glass plates are temporary sealed.

Seperation gel is poured first after polymerisation of the seperating gel. then tacking gel is poured.

Samples are loaded onto a gel of polyacylamide which is placed in electrophoresis. When the voltageis applied -ve charged molecule goes to anode in gel. Proteins are blotted on PVDF membrane.

The lane in which buffer A was added in the sample will give two band one at 30 K DA and 40 K DA.

in Second buffer B was added sampkle will give one band of 70 K DA This happens because of breakage of disulphide bond.

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