Removing annoying heavy chain detection in IP/blots

Frank O. Fackelmayer Frank at Fackelmayer.de
Thu Aug 22 07:35:43 EST 2002


 > take longer than a minute or two), repeat once, then neutralize by
> > adding 1ml of 500mM Tris pH8. Let drop through, and equilibrate the
> > material with your buffer of choice, e.g. PBS.
> 
> Thanks for the suggestion.  We never considered going down as far as pH 2.0
> !  My understanding was that at pH 3.0 glycine the Ab stays bound to Protein
> A.  So your point is very helpful here.  I will give it a try.  I presume
> you meant to wash the beads this way BEFORE IP. Then IP as normal.  

yes.

> However
> I am still worried that the Ab may be damaged by this pH (afer all, it has
> just gone thru a cross-linking exercise too).

we routinely purify antibodies with exactly this buffer, and never got
problems. As Dima suggested, there are other "high stringency" buffers
you could try as well.

> > supernatant behind), add 300ul of Methanol, vortex, spin again, remove
> > all supernatant (a pellet may not be visible), air dry pellet for at
> > least 30min, redissolve in sample buffer and analyse on a gel.
> 
> Now we are talking about getting the antigen off the beads, leaving the IgG
> behind if possible.  If you are right, that both SDS and DTT treatments of
> the beads pulls off some IgG, then that may be the biggest part of our
> initial problem (ie detecting unwanted trace HC and LC in the subsequent
> blot), rather than inefficient crosslinking as we initially thought. 

yes, most probably. Crosslinking with DMP is quite efficient in our
hands, but there certainly remain non-covalently bound IgGs that you
should wash away. 

> Are
> you saying that to avoid the presence of trace eluted Ab, the glycine
> elution (or the 3.5 M MgCl elution suggested by DK (thanks)) will have less
> or no IgG contamination, compared with heating the beads in SDS or treating
> with DTT?

yes. This is because you elute the antigen with the same buffer you used
for washing the sepharose before. All IgGs that can come off already did.


> I suspect this idea will help, but is much more work because the elution
> volumes are larger than simple SDS.  To concentrate the eluates I routinely
> use the Wessel & Flugge method you suggest, myself.  [Wessel,D. &
> Flugge,U.I. (1984) A method for the quantitative recovery of protein in
> dilute solution in the presence of detergents and lipids. Anal.Biochem. 138,
> 141-143].  It is the most powerful and simple one that I know! 

that´s why I suggested it :)

> However, I
> find that more than half the people I show/teach it to cannot get it to work
> properly or reproducibly.  
> There is nothing wrong with the method, but it
> requires a lot more attention to detail than a lot of people apparently give
> their experiments. 

try to keep persons out of your lab that do not care about their
experiments. :)

> The last step is always the problem, making sure the
> pellet dissolves in the SDS. We find we have to sonicate with a fine probe -
> messy, and the SDS foams up and has to be centrifuged down to de-foam before
> gel-loading.

redissolving in sample buffer is a problem with all protein
precipitation methods. Compared to TCA, e.g., the Wessel/Fluegge method
is much less of a problem. Do not overdry your samples!
You are right, of course, that you have to redissolve the pellet
properly. When there is a lot of protein, sonication helps a lot, as you
said. In addition, we spin the samples in a microfuge for a couple
minutes before loading the gel when we see that the pellet is hard to dissolve.


> 
> > > - Alternatively, has anybody ever tried this idea?  Incubate protein A
> beads
> > > with antibodies (don't chemically cross link).  Do the IP, then elute
> all
> > > off the beads with SDS but no mercaptoethanol.  Then run the gel without
> > > mercaptoethanol either.  In this way, the IgG should remian as a 160kDa
> > > complex, leaving only nice clean target antigen/proteins to be detected
> in
> > > the 55 kDa region of the blot.  A nice idea, but does it work?
> 
> > Yes, it will work. However, not treating the sample with mercaptoethanol
> > will result in aberrant mobility of many proteins, because they contain
> > SS-bridges that will remain and keep the protein in a constrained
> > conformation. If your protein does not contain SS-bridges, this is
> > irrelevant, of course. Just keep in mind that certain marker proteins
> > may not run as expected...
> 
> Thanks again.  Yes we are aware of the problems associated with this idea.
> However in our case we are lucky.  All our target proteins for the IP are
> monomeric and do not form S-S bonds.  We explored this earlier.  So we will
> readily see our target protein OK.  As you point out, we may miss some of
> the proteins that co-IP with our target, but we don't want them at this
> stage.
> Have you (anyone?) actually tried this idea?  Do you actually see the IgG
> migrating at 160-175 kDa when heated in the absence of betaME?  

yes, we did. Otherwise I wouldn´t say it works...

> Is there
> actually no HC at 55 kDa left?  We don't want even a trace amount of HC.

I haven´t looked at "trace amounts", but it did not disturb. Much less
at least than protein A that leaches from the sepharose (and also runs
at that mol.wt. range) under many conditions. You´ll easily see it on
your western blots with lanes containing control IPs with no antibody...


best wishes for your experiments,
Frank



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