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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Replies
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We have used phospho-p38 antibodies from Cell Signaling, and the results are pretty clean. Although we have similar background problems with regard to phospho-JNK because it is somewhat hard to detect.
What are you using to lyse your cells? Are you including protease inhibitors? Phosphatase inhibitors?
We use 5% BSA in our primary antibody dilution buffer and 1% BSA in the secondary, that may help. I’ve also tried 1% milk with the secondary stain in order to reduce background.
Hope this helps.
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Hi Brock,
Are you using phospho-p38 from cell signaling catalog #9216?
I make my own lysis buffer and I add leupeptin and aprotinin, NaF is also included, as well as Na3VO4 and Na4P2O7.
I use 1% BSA in my primary and secondary antibodies.Thanks! Fabiano
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Brock,
Just some additional questions.
I´ve been trying this experiment for weeks.
What´s your lysis buffer?
Which phosphatase/protease inhibitors do you add?
What´s your primary antibody working concentration? I use 5 microliters for a final volume of 10ml.
Thanks again, Fabiano -
Hallo everybody,
I cannot help directly since used to have the same problem as Fabiano some time ago (> 3 years). I did not follow up. I remember that Margot Thome at University Lausanne was experienced with anti-phospho-p38 antibody. She is very nice person and will help surely: Margot.ThomeMiazza@unil.ch
Good luck!
Marek -
The phospho-p38 antibody we use is #9211S.
I have used RIPA and Buffer A for lysis reagents. I supplement these buffers with:
500uMDTT from 1M stock,
1.25mM PMSF from 100mM stock,
PIC Tablets (Roche 11873580001) 1 tablet into 500ul dH2O = 100X stock, PPi cocktail (50X stock = 50mM NaPPi, 50mM NaV3O4, 50mM NaF, 500uM Na2MoO4.I use the antibody at a 1:1000 dilution, 2ul into 2ml using heat-sealed bags and stain either overnight at 4deg or 1 hour at RT, both work well.
Hope this helps
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Thanks a lot! This is a very nice forum and you’ re helping me a lot. I’m a post-doc working in San Diego. I had my PhD in France. I’m fascinated for cell signaling and bacterial infections.
I’ll try to put my primary antibody in BSA5% and if it doesn’t work, I’ll change my lysis buffer for Brock’s one.
Brock,
Many thanks. Where do you keep your stocks of DTT, PMSF, PIC and the PPi cocktail? At 4 or -20 degrees? Is this PPi cocktail prepared by you or you buy it already prepared?
Marek, thanks for your help.
Nice to meet you all. Fabiano -
Hello everybody,
I tried my primary antibody in BSA5% and there’s still some background and inespecific bands.
I’m purchasing the reagents to try to optimize my lysis buffer, as suggested by Brock.
I’m in doubt, because RIPA contains EDTA and some serine proteases depend on metals to work well.
Brock,
Don’t you think the Roche tablets can be inactivated by the EDTA in the RIPA buffer? Do you keep the protease and phosphatase inhibitors at 4 or -20 degrees?
Sorry if I ask to many details, but I want to understand what I’m doing, because I want it works and you seem to have a good experience on it.Merry Christmas,
Fabiano
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Fabiano,
Sorry for the delay. I keep the cocktails of inhibitors at -20C in aliquots. The PPi is made from scratch in the lab using the list of chemicals I posted. Then this cocktail is aliquoted and stored at -20C.
As far as EDTA inhibiting serine proteases, I think you would want that. The point of adding these inhibitors is to halt protease and phosphatase activity.
If you are concerned with using RIPA buffer, you can also try the Buffer A, but it will not lyse the nuclei, which may not matter for you since you are looking at cytoplasmic proteins and may actually clean up your prep.
The recipe for Buffer A: 10mM HEPES pH 7.9, 10mM KCl. 0.1mM EDTA pH 8, 0.1mM EGTA, optional (0.4% Igepal/NP-40). As you can see, this buffer contains EDTA, but a low concentration, but I haven’t had any proplems using these buffers to look at phosphorylated cytoplasmic proteins.
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Brock,
I misunderstood.
I thought that metals could be important for protease inhibitors, but they’re actually important for proteases function, so EDTA might be important for the buffer.
Your answer is very clear.Thanks again,
Fabiano
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Hi everybody,
I contacted Margot Thome and she told me that she gave up trying to blot phospho-p38, because it doen’s work well. If she had to do it again, she told me that she would immunoprecipitate p38, but unfortunatelly there’s no good antibody for this.
As Brock seems to have a good protocol and many articles show nice results, I believe i’m just having technical problems.
I tryed Brock lysis buffer yesterday, but the signal was very weak (only a shadow after 20 minutes exposure), although now my blots are much cleaner and without inespecific bands.
I wonder if it’s not a problem related to my NaPPi cocktail (the same Brock uses), because I found really difficult to dissolve the NaPPi (I had to vortex a lot to dissolve the powder and it precipitates again after thawing).
Brock, do you have a similar problem?I’m trying the blot again today, increasing the concentration of primary antibody from 1:2,000 to 1: 1,0000 (in BSA 4%) and using the double amount of secondary.
I would appreciate to receive Brock comments once more. Thanks Marek for your help.
Fabiano
Results
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