serum protein delipidation question

[Jeffrey W. Chisholm]

In article <3k506i$n1n at dove.nist.gov>, dmb618 at enh.nist.gov says...
>
>Does anyone have any "tried-and-true" methods (or 
>references to methods) for defatting serum proteins?
>
>I've been trying to isolate albumin from serum on a 
>microscale using antibody affinity chromatography.  
>While I have been able to extract the albumin, the 
>albumin retains its complement of bound small 
>lipophilic molecules (which interfere with the 
>quantitation I ultimately want to do).
>
>I have tried to defat the extracted albumin using the 
>standard free fatty acid extraction solvent of 
>chloroform/MeOH (2:1).  The problem here is that the 
>MeOH precipitates the protein which settles on the 
>chloroform-water interface, making it difficult to 
>recover.
>
The standard technique for delipidating serum apoproteins uses a series 
of ethanol-ether extractions at -20 C and can be found in

"A guidebook to lipoprotein technique", Vol. 14 of Laboratory 
Techniques in Biochemistry and Molecular Biology.  Mills, G.L, Lane P.A. 
and Weech, P.K., pg. 469 (1984)

However, I don't know how efficient this technique is in removing fatty 
acids and the procedure is rather large scale.  I am currently in the 
process, actually I started this afternoon of adapting the process to 
microscale (ug quantities of protein) and should have answers in a couple 
of days. I need to free albumin from the interior of lipid vessicles so 
that I can immunoquantitate it and I certainly hope my antibodies are not 
affected by bound fatty acids.

However, you might also check the Sigma Cataloque as they reference a 
method in JBC that they use for the preparation of F.A. Free Albumin.  
This is probably a large scale procedure, but you might be able to scale 
it down.

BTW, if you can purify the albumin using your anti-albumin antibody, why 
can't you immunoquantitate it against commercially prepared albumin that 
hasn't been delipidated.  This will work as long as you are reasonably 
sure your stds. are comparable to your samples.

Hope this is of some help,

Jeffrey W. Chisholm
Lipoprotein Research Group
Dept. of Biochemistry
Dalhousie University
Halifax, Nova Scotia, Ca.

Email: JWChish at ac.dal.ca