Question

1). A mixture of proteins was seperated on a sizing column. one 2ML fraction has two proteins left in it,and the technician wants to run an ion-exchange coloumn. First, he or she has to dialyze it into the appropriate buffer. how much ion-exchange buffer is needed for dialysis?

2). A technician sets up a negatively charged ion-exchange column and puts a small volume of a mixture of the proteins on it. what should happen to positively charged proteins in the mixture? what should happen to negatively charged molecules in the mixture?

3). As a sample elutes from a column, the technician wants to determine the concentratioj of protein in the sample. what instrument can be used to determine the concentration of the sample and at what setting(s) should it be operated?

Answer 1

1. (ANSWER & EXPLANATION)

Dialysis - is a technique used for the separation of small molecules and proteins using a semi-permeable membrane. It is probably the first and oldest method of buffer exchange. The dialysis bag is filled with a concentrated solution of proteins and the molecules that are small enough to pass through the pores of the bag, move into the dialysate (or buffer solution). The movement of molecules is from high concentration to lower concentration.

The volume of buffer to be used for dialysis is 200 to 500 times the sample volume. In this case, as the volume of the sample is 2ml, the volume of the solution required for dialysis would be 400ml to 1000ml.

2. (ANSWER & EXPLANATION)

A negatively charged ion-exchange column will attract the positively charged groups whereas repelling the negatively charged groups.

When positively charged proteins are made to pass-through the negatively charged column, they will be attracted as the groups have opposite charges and are bound by ionic interaction.  

When negatively charged proteins are made to pass-through the negatively charged column, they will be repelled as the groups have similar charges and would be eluted out from the column.

3. (ANSWER & EXPLANATION)

The instrument that can be used for the estimation of protein concentration is a Spectrophotometer in which absorbance is measured at 280nm.