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.

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

 

well it sounds like an elimination process from blood serum to me, as for the 1`st step, it`s an affinity chromo technique for Hydrophobic, then it gets rid of the Hydrophilic parts.

 

it`s not my area of Science though