how to concentrate Co-IP protein sample
PC at nospam.edu
Thu Mar 18 00:23:26 EST 2004
higher concentration of SDS in your sample will normally not influence
quality of PAGE separation. You are going to run MS on the in-gel
digested samples - why worry then?
It is strange that pH2.5 was not enough to elute your protein. It might
be that your protein is not soluble in acid (too hydrophobic? low pI)??
You can always try elution with 3M KSCN - another very good method. I
believe it is compatible with TCA (I would still try it on something
cheap like RNase or lysozyme first).
I have recently seen a protocol where they incubate anti-HA beads with
1mg/ml HA peptide at 37C (!) for elution - sounds not very promising. In
that respect anti-FLAG M2 Ab is much better.
In any case, what you normally want to do is to reduce leaking of the Ab
from the beads. If you heat your sample in SDS buffer, even without DTT,
you cannot avoid it. This is a problem especially when your specific
bands are weak.
I would also first try to do IP from cells metabolically labeled with
35S and see if there are any proteins co-IP with yours.
Extraction/buffer conditions are important and can be easily optimized
in this way.
BTW, check this link (paste it in one line):
> Thank you very much for your reply.
> My final goal is to identify some novel proteins that come down with this
> HA-tagged protein using mass spectromety.
> Do you know if excessive SDS will inhibit mass spec analysis?
> I have tried eluting the protein with glycine (pH2.5), but at least on the
> western, almost none of the protein got eluted. I don't why, I just simply
> 100ul of glycine to about 20ul of HA-beads, let it sit for about 5 minutes,
> then spun down the beads and got the eluate. Is it the right way to do it?
> "P.C." <PC at nospam.edu> wrote in message
> news:c35qmv$1if$1 at cruncher.dfci.harvard.edu...
>>Microcon-3 (from Millipore) should be just fine (you will concentrate
>>SDS too to some extent, but that does not matter too much).
>>You could also elute the protein in acid (Gly-HCl, pH2), and then do TCA
>>(may be first neutralize HCl would be safer (?)).
>>On the other hand, it might be more efficient to upscale and use more
>>concentrated extract in your IP with same amount of Ab. Did you check by
>>western if you IP most of your HA-tagged protein?
>>>I am trying to do a co-IP experiment on a HA tagged protein, and I
>>>the protein with SDS buffer. However, the protein level is very low
>>>after I ran the eluate on a gel followed by silver staining, I couldn't
>>>any band. So now I am thinking combining all the eluate from multiple
>>>experiments. Is there a way that I can concentrate all these proteins?
>>>Will TCA precipitation work in the presence of SDS? Or is there any
> kind of
>>>concentrator that I can use? Thank you very much!
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