Question

how to calculate affinity column recovery and ion exchange recovery in LDH lab?

Answer 1

1. Affinity Chromatography

This technique uses the advantage of the high affinity of many proteins for specific chemical groups. Affinity chromatography is used for isolating transcription factors, proteins that regulate gene expression by binding to specific DNA sequences. A protein mixture is percolated through a column containing specific DNA sequences attached to a matrix; proteins with a high affinity for the sequence will bind and be retained. In this instance, the transcription factor is released by washing with a solution containing a high concentration of salt. In general, affinity chromatography can be effectively used to isolate a protein that recognizes group X by (1) covalently attaching X or a derivative of it to a column, (2) adding a mixture of proteins to this column, which is then washed with buffer to remove unbound proteins, and (3) eluting the desired protein by adding a high concentration of a soluble form of X or altering the conditions to decrease binding affinity.

2.  ion exchange Chromatography

If a protein has a net positive charge at pH 7, it will usually bind to a column of beads containing carboxylate groups, whereas a negatively charged protein will not. A positively charged protein bound to such a column can then be eluted (released) by increasing the concentration of sodium chloride or another salt in the eluting buffer because sodium ions compete with positively charged groups on the protein for binding to the column. Proteins that have a low density of net positive charge will tend to emerge first, followed by those having a higher charge density. It separates proteins based on their charges, using a column containing the positively charged anion exchanger Q-sepharose agarose (the solid state), and a buffer (the fluid state), which can include a salt.  Negatively charged lactate dehydrogenase adheres to the positively charged Q-sepharose, and will not elute (come out of the column) until it is displaced by NaCl.