Mr of Human Serum Proteins by SDS-PAGE

Dima Klenchin klenchin at REMOVE_TO_REPLY.facstaff.wisc.edu
Mon Feb 26 20:04:59 EST 2001


chriselk at med.unc.edu (Chris Elkins) wrote:
:Dear Sir/madam,
:    I am a bacteriologist working on the binding of serum proteins to my
:
:bacterium.  My assay consists of iodinating NHS, binding the labeled
:serum to bacteria and isogenic mutants and see what proteins bind to the
:
:parent but not the isogenic bacterial mutant.  After binding of NHS to
:bacteria and washing, I run SDS-PAGE gels and do autoradiography.  I
:have identified a
:number of hot bands.  I would like to look up somewhere what these
:proteins might be based on Mr by PAGE, and then confirm with
:immunoblotting, etc.  Remember that I cannot sequence these directly,
:since the bacterial proteins are present and they iodinated serum minor.
:
:    Do you know of a reference where I could find the molecular weight
:of serum proteins by SDS-PAGE?
:

Sorry, I don't know one that's complete enough for your purposes.
Since serum contains easily over 100 proteins, your guessing 
approach does not promise to be very exact and productive. 
Here is the one I'd use:

1. Extract parent bacteria with 1-2 M NaCl (or whatever does not
lyse/permeabilize them), concentrate proteins in the supernatant, 
immunize couple rabbits with the mixture. 
2. Purify IgG from immune sera and immobilize them at ~ 5 mg/ml
on Sepharose.
3. Add serum to the _excess_ of parent strain, wash, elute
bound proteins with high salt, collect sup, dialyze into PBS.
4. Add sup to the _excess_ of isogenic strain bacteria, incubate, 
spin => sup contains proteins that did not bind. 
5. Since many of the proteins in the sup will be extracted
bacterial proteins, run the protein mixture over 
_excess_ of anti-bacterial IgG column.
5. Presto! Most of the proteins you now have in flow-through 
are the ones that bind parent but not isogenic strain. Concentrate
by precipitation, run gel, excize bands and sequence directly
on mass-spec. 

I'd be interested to learn what might be wrong with such an 
approach. 

HTH,

        - Dima






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