serum protein delipidation question

Chris Birkett chris.birkett at bbsrc.ac.uk
Thu Mar 23 12:28:09 EST 1995


> 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 charcoal method of Chen might be what you're looking for.

Chen R. F. (1967) Removal of free fatty acids
from serum albumin by charcoal treatment.
J. Biol. Chem. Vol 242 pp 173-181.

I used to use this for preparing FFA-free albumin quite frequently.
As far as I know it does not damage the protein's ability
to bind FFA or other lipophilic moieties, my own application was
to bind tritiated FFA for feeding to cells as a metabolic tracer.
I don't know how easy it would be to work at the micro-scale you
require or exactly what you need to quantify, but
I expect that you could use a microspin column of one sort
or another.

The method in it's essentials is goes as follows:  mix aqueous slurry
of activated charcoal with albumin solution, carefully 
acidify to pH 4.0, stand for 60 mins. in ice, spin out the charcoal
then neutralise the supernatant (containing the albumin).

Hope it might be useful.

Christopher R. Birkett
IAH Compton Laboratory
Compton, Nr. Newbury,
Berkshire, RG16 0NN, UK.

E-mail:  chris.birkett at bbsrc.ac.uk





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