Suppose you have a mixture of five proteins listed in the table below: Indicate

Question

Suppose you have a mixture of five proteins listed in the table below: Indicate the order in which there proteins elute from a Bio-Gel p-10 gel-filtration column (starting with the on that elutes first). Is this the best resin to separate all the components of this mixture? Why? If not, what other Bio-Gel resin can you use to separate all the components of the mixture? This requires you look up some information about the size exclusion limits for each type of resin You load this mixture on a cation exchange column. The buffer you use for this column is acetate buffer, pH 4.76. List the protein or proteins that will appear in the flow through (will not bind to the column) In order to elute those proteins that are immobilized on the column, you then apply a linear salt gradient, with NaCl concentration gradually increasing from 0 to 1M. Indicate the order in which the proteins bound to the column will elute as the salt concentration increases. Repeat the same procedure as in b. But now you use anion exchange column, and the buffer is TRIS (pH8). Answer the same questions as seen in i. and i i.

Explanation / Answer

a) Gel filteration is used for separation of proteins based on their molecular weight or size

The protein with smaller size fits in side the pores of gel and the protein with larger size elute first

so here in the given set of proteins the elution order (starting with the protein which will elute first) will be

Protein 1 , Protein 3, Protein 5, Protein 4 and lastly Protein 2

b) Here we are using acidic buffer with pH less than 7.

Protein with pI < 4.76, will carry negative charge.

Protein with pI > 4.76 will carry positive charge.

We have taken cation exchange resin which is negatively charged.

so the proetins which are positively charged will bind to the resin

Here all except protein 5 will bind to the resin, rest all will flow.

The order after adding NaCl will be

Protein 3 , 2, 4 ,1

c)

Here we are using acidic buffer with pH = 8.

Protein with pI <8, will carry negative charge.

Protein with pI > 8 will carry positive charge.

We have taken anion exchange resin which is positively charged.

so the proetins which are negatively charged will bind to the resin

Here all except protein 3 will bind to the resin, rest all will flow.

The order after adding NaCl will be

Protein 5 , 1 , 4 and 6

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