4. In the laboratory, we used gel electrophoresis to compare three forms of ribo

Question

4. In the laboratory, we used gel electrophoresis to compare three forms of ribonuclease A (RNAse A) . The native proteirn . The protein with its disulfides reduced and the thiols modified with iodoacetic acid (RCM) . The protein with its disulfides reduced and the thiols modified with iodoacetamide (RCAM) Suppose that you were to subject these three samples to gel-permeation chromatography Assume that you use a chromatography matrix for which Kae lies between 0 and1 for all three forms. Be sure to explain your reasoning in answering each of the following questions: Which form (or forms) of RNAse A would you expect to elute from the column first? For which form (or forms) would you expect Kave to be largest? Suppose that you wanted to separate the native and RCAM forms of RNAse A by affinity chromatography. Suggest a type of molecule that you might attach to a chromatography matrix that would enable you to specifically bind one of the two forms to the matrix. Which form would be bound? Suggest a chromatographic method that could be used to separate the RCAM and RCM forms of RNAse A. Briefly explain how this method would separate the two forms

Explanation / Answer

we know that in gel permeation chromatography the largest molecule or peptide is eluted first. later comes the molecule of less size. Here the native molecule is about 13000 to 15000 kd. when it is treated with the iodoacetamide , the histidine residues are alkylated. hence we can understand that the molecule is much more larger than the native RNAase. hence the elution of the Rnase follows in this way RCAM , RCM then comes the native form.

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