8. Protein purification. Identify the best chromatography for purifying the foll

Question

8. Protein purification. Identify the best chromatography for purifying the following pairs of polypeptides. [In the spaces provided: i write the acronym of the best chromatography resin: DEAE (diethylaminoethyl), CM carboxymethyl), or GF (gel-filtration); and ii) indicate which polypeptide moves more slowly through your chosen resin at pH 7.4.] A) Polypeptides: #1:ACDEFGHI and #2: KLMNPQRS Best chromatography resin: slower moving polypeptide [Write #1 or #2.] Polypeptides: Best chromatography resin: slower moving polypeptide [Write #1 or #2.] B) #1: STILL and #2:WATER C) Polypeptides: #1: AFGI and #2:LMNPQVwY Best chromatography resin: slower moving polypeptide [Write #1 or #2.] -

Explanation / Answer

Diethylaminoethyl (DEAE) is a resin i.e. positively charged and used in ion-exchange chromatography. DEAE resin binds with negatively charged proteins into the matrix. The bind proteins then released by increasing the solvent salt concentration or changing the pH of the solution. its functional group is -N+(C2H5)2 and have pH range 5–9.

Carboxymethyl (CM) resin is a weak cationic exchanger having functional group -COO- with pH range 5–9.

Gel filtration chromatography (GF) sparates protein on the basis of their size, shape and weight. Smaller peptides diffuses into the pores of the beads, hence it moves slowly as compared to larger peptides.

A) best chromatographic resin may be used either DEAE or CM. If DEAE resin is used then 1st peptide move slower because of net negative charge on polypeptide due to the presence of aspartic acid and glutamic acid. if CM resin is used then peptide 2 moves slower because it has positive charge due to the presence of lysine and arginine.

C) best chromatographic resin may be used are GF resin. peptide 1 moves slow through this resin because of small size.

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