phosphorylation in cell lysates
Fabiano Pinheiro da Silva
Wednesday, 12 December 2007 04:38 UTC
Hi, I´m trying to see p38 activation using anti -phospho-p38 antibody in cell lysates, but there´s too much background and many bands. p38 appears as a weak and doubtful band.
Is this the appropriate way to investigate phosphorylation of proteins?
I´ve seen some publications using similar techniques, but I´m in doubt if I can trust in my results.
Maybe I should try IP p38 to have a clear result, although using cell lysates it´s easier and I “can” dissect a pathway faster.
Does anybody have suggestions? What do you do to have black and white results in cell lysates?
My actin control is very nice, but when I try phospho-proteins there´s a lot of background.
Thanks for your help, Fabiano
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Hi Brock,
How do you strip your membranes?
What stripping solution do you use?Thanks, Fabiano
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Hi everybody,
just one more question: some precipitates are appearing in my samples during lysis.
Does anybody ever had a similar problem?
I had the same problem using two different buffers, although in both I added aliquots of PMSF, PIC and phosphatase inhibitors from the same source.
I’m still using the same lysis buffer protocol that worked well many times. Are my reagents getting old?
Can you give me any suggestion? I’m trying not to change all over again.
Thanks, Fabiano -
Fabiano,
The stripping buffer I use is RESTORE Western Blot Stripping Buffer from Thermo Scientific #21059
I wash the blot with TBS-T after development, add ~20ml (just enough to cover the blot) and incubate in an agitating water bath at 37 degrees for 15 minutes followed by additional washes with TBS-T. It is necessary to block again (I usually do 30-60 min with 5% Milk + TBS-T) before primary staining.
Good luck.
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I’ve had pretty good success with phospho-p38. I use Cell Signaling #9211S.
I’d recommend just using cytoplasmic extract instead of whole cell extract (this strategy can clean up phospho blots, but also gives you the opportunity to screen nuclear extracts for Rel subunit translocation or perform gel-shift assays). Use the Buffer A lysis reagent I described in a previous thread.
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Thanks Brock, it’s always great to hear your comments.
Shying, I think you misunderstood.
RIPA buffer classically contains NP-40, although you can find modified RIPA with triton.
NP-40 is an ionic detergent and must be avoided for IP purposes, because it disrupts protein-protein interactions. use non-ionic detergents. i suggest start by using 0.5% triton if you want to co-immmunoprecipitate proteins. -
Hallo everybody.
Thanks guys for great discussion!
Ad NP-40:
NP-40 and Triton X-100 are both mild non-ionic detergents and are widely used used as a replacement of each other. Both will solubilize (not completely) plasma membrane but will leave nuclear membrane relatively intact.
There is some confusion around with NP-40 (see Wiki pages) therefore it is safer to use Triton X-100.The useful pages:
NP-40
Triton X-100Best,
Marek -
Thanks Marek for the correction.
NP-40 is an non-ionic detergent.
I should have confirmed that better, my concept was wrong, although I´ve never seen co-immunoprecipitations work with NP-40 (it seems too strong, but it´s just my personal experience).
The problem with RIPA is that it contains sodium deoxycholate, which is an ionic detergent.
Thanks again, Fabiano
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