In my biochemistry lab, were purified Lactate Dehydrogenase from chicken meat. T

Question

In my biochemistry lab, were purified Lactate Dehydrogenase from chicken meat. The protein was extracted by use of 60% NH4SO4 and then dialysis as performed. The protein was isolated by affinity chromatography with a cibracon blue column. The column was done by collecting 5 ml fractions with the following washes: TRIS buffer, NAD solution, Tris buffer, NADH, and tris buffer. The protein was found to be colelcted in the NADH fractions. My two questions are as follows:

Why was the removal of ammonium sulfate necessary for purification?

And how does NADH help to elute the protein from the column?

Explanation / Answer

1. It is important to remove ammonium sulphate for purification of Lactate dehydrogenase because it will interfere with the purification process..Ammonium sulphate might compete with LDH for binding to the column. Therefore it is important to remove ammonium sulphate by dialysis.

2. During affinity chromatography, NADH is used to bind Lactate Dehydrogenase (LDH) to the cibracon blue column. NADH acts as a ligand and results in the formation of LDH-NADH complex which binds to the cibracon blue column. As long as NADH is present in the washing buffer, LDH will remain adsorbed to the column.

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