Protein immobilization on Sepharose using periodate activation (Was: Purifying Antibodies using affinity support)

immunechem immune at intergate.bc.ca
Thu Mar 11 14:53:02 EST 1999


Here I am telling you that Periodate method is one of the most 
inefficient coupling procedures, particularly for the protein sample 
with low concentration. 

I am suggesting some of the direct, one-step and simple but highly 
efficient activated matrices comercialy available.
1.	Activated ECH Agarose. Available from both Peirce and Sigma. 
Providing 6-carbon spacer and an active NHS ester as leaving group and 
form stable amide linkage with your amine containing protein. 
2.	Cynogen Bromide activated Sepharose. One of the most efficient 
matrix available and provide one carbon spacer but has very bad 
reputation for leaking and ionic exchange effects.
3.	A novel activated matrix developed by 3M and sold through Peirce 
Chemical. The matrix contains a active lactone, which is readily form a 
stable amide with primary amines of the protein. Disavantage: hyarolysed 
lactone create a free carboxyl=ionic effect; low flow rate of its 
acrylamide polymer matrix.
4.	Activated thiol Agarose. Available from Sigma. It forms -S-S- 
bond with free thiol (cysteine residue) of the protein effiently with 
high yield (the matrix is not sensitive to water as others). 
Disavantage: you need to reduce your protein with DTT first before 
coupling.
5.	Iodoactate activated Agarose. Available from Peirce. High yield 
and stable for free thiol containing protein. 

The disavantage of Periodate activated Agarose: need high pH (>pH9) to 
obtain a good high yield and efficient; Almost no good for protein less 
than 0.1 mg/mL. The formation of Shif-base is not reversible unless 
furthewr reduced to secondary amine (harmful to lots of proteins). 
Oxidation damage the matrix and create leaking problems. 

Beside, I have to warn the one who put the first question: affinity 
purification of antibody using whole protein will create lots of 
problems and limits the application of the antibodies because of the 
leaking of antigen, WHICH CONTAINS MULTI-EPIOPES. Unlike eptope-based 
affinity chromatography, the leaking antigen will interfere the 
interpretation of your results.



>I checked with Dima, and he clarified something for me.  He means to 
say
>that you must use the CL-4B, which is cross-linked, because plain 4B, 
which
>is not cross linked, will not work with this activation method.
>
>Thanks Dima.
>
>Warren Gallin
>Department of Biological Sciences
>University of Alberta
>Edmonton,  Alberta     T6G 2E9
>Canada
>wgallin at gpu.srv.ualberta.ca




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