Purification of cyclooxygenase (COX)

[6233]

I'm doing a project on the purification of sheep platelet cyclooxygenase and have read a paper. But there are some steps of the protocol which I don't understand.

First, hydrophobic chromatography using ibuprofen-sepharose affinity column was used. Next, fractions were subjected to 2 metal-chelate chromatographies - IDA/Zn column then TED/Zn column. Cyclooxygenase binded to IDA/Zn which was eluted with imidazole. The cyclooxygenase-containing fractions were put through the TED/Zn column but cyclooxygenase did not bind and was collected in the unbound effluent.

The unbound effluent was then put through a Haemin-Sepharose affinity chromatography column and purified cyclooxygenase was obtained.

Can anyone pls enlighten me about why a series of affinity chromatography was used and what proteins were gotten rid of in each step?

IDA= iminodiacetic acid; TED= Tris(carboxymethyl)ethylenediamine.

Thanks.

[molecule2005]

Hey there 6233,

Protein Purification is an art more than a science. It is usually a set of trial and error methods.

ie the investigators tried to separate COX using various methods. They looked at the fractions and assessed which conditions were collecting COX into neat fractions.

What proteins are removed at each step? This is a very difficult yet easy answer. Most proteins from the cell fractions would be removed that have properties different from COX, and that would be many!

Exactly which proteins? very tough, as you need to do proteomics to see all the proteins remoed. Obviously COX would not be removed at these steps, thus the best purification is to remove EVERY protein EXCEPT COX.

The investigators needed to visualize every fraction to see COX is not removed.