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IP Problems


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#1 stressed

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Posted 24 February 2009 - 10:58 AM

Hi there

I am struggling with an immunoprecipitation. I use an M2 flagtag antibody from Sigma to IP for a membrane protein, but I cannot detect my protein on a blot.
This is my protocol:
I transfect 1000 000 cells per plate and allow them to grow for 48hrs.
the cells are harvested in 200ul of 1% triton-x and incubated on ice for 45mins (protease inhibitors).
i then centrifuge my whole cell lysate and use the supernatant for the IP.
800-1000ug of protein is incubated with the Flag M2 antibody for 2hrs (antigen antibody compex).
i thereafter incubate the antigen-antibody complex with washed protein G sepharose beads
2hrs-overnight
the sample is eluted from the beads after boiling in 5xLaemmli buffer at 100C for5-10mins.
i then run my sample overnight on a large 12% gel.

PROBLEM:
i can clearly see the light chain but it is a bit smeared and much darker than the heavy chain, but i dont detect the antigen.
Infact the blot seems to present with lower bands just below the light chain. This is odd as it appears as if the antibody is co precipitating a non specific band throughout the blot (around 23 kDa) or could this be a product of the antibody?
Could this be due to the use of sepharose G beads which expired in 2000? The beads have been kept at 4C.
could they over time have dissociated and therefore interfere with the binding of the antibody?
or could it be that because I have not added protease inhibitors to the antigen-antibody complex and this resulted in a breakdown of my antibody?

Any ideas would be appreciated! :)

#2 Dr Teeth

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Posted 24 February 2009 - 12:34 PM

Your procedure seems appropriate to me. I do IPs quite often using Sigma's FLAG (M2) antibody and the M2 is highly sensitive.
A few questions:
1) Can you see your FLAG-tagged protein of interest in the input lanes on your Western?
If not, are you sure your FLAG-tagged protein is being expressed properly?
If so, does it appear to be highly expressed? If not, try increasing your input to 2 mg.
2) Have you tried using the Sigma M2-agarose beads for IP?
These beads have M2 antibody pre-conjugated, so there is no need for protein G and they work quite well.
3) Are you confident in your protein transfer from your gel to the membrane?

Science is simply common sense at its best that is rigidly accurate in observation and merciless to fallacy in logic.
Thomas Henry Huxley

#3 Curtis

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Posted 04 March 2009 - 09:02 PM

1000ug seems to be enough but I often go for more than 2000ug of total lysate

I bind the antibody to protein G first before adding the lysate. this gives me better results, this is a method that I learned from many people on this forum. we do not add the antibody directly to the lysate. we usually bind the antibody to protein G (1-4 hours end-to-end rotation at 4 degrees), and then block it with filtered 1%BSA in PBS for 1-2 hrs.

I get pretty sharp bands on my blots this way. my protein stay 3-4 KD below the light chain on a 15% gel.




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