Edited by mdfenko, 20 October 2010 - 06:38 AM.
Western blot fail, time and time again
Posted 20 October 2010 - 06:38 AM
genius does what it must
i do what i get paid to do
Posted 20 October 2010 - 07:29 AM
Hey Guys~ I'm a little confused. Doesn't the fact that he's getting signal in the western blot with a positive control lysate (I assume commercially obtained) mean that the antibody is sufficient for western blot and indicate that the problem is in his sample preparation?
I agree with mdfenko -- it sounds like denaturing your protein results in the loss of the epitope(s) your antibody recognizes. Is this a monoclonal Ab, or are you using polyclonal sera? Try a dot-blot of your samples and see if your Ab recognizes them if they're not denatured (just spot a small amount of each along with a positive and negative control on a suitable membrane, and process the membrane as you would a western).
Posted 20 October 2010 - 10:18 PM
I`m now using a home-made lysis buffer with NaCl, EDTA,Tris and protease inhibitors and 10% Glycerine.
Can I also use RIPA buffer (Sigma) with protease inhibitors for the sample preparation?
Posted 21 October 2010 - 08:41 AM
Posted 22 October 2010 - 04:23 AM
One where I used my protein samples and one with my sample+ DTT loading buffer (so reduced)
In the first blot with only the native protein I did not get any signal (also not from my positive control)
In the second blot with the reduced protein my positive control gave hell of lot signal and non from my samples.
So conclusion: the Ab picks up the reduced form of the protein and in my samples there is no protein detectable?
The problem is that the protein i`m trying to detect is phospho-SMAD. I used TBS, I used protease inhibitors but I suspect
that I lost the phosphorylation somewhere.
I`m now going to try another antibody (not phospho) + beta-actin staining on the samples and hope to see some signal there.
Anyone still any suggestions?
Posted 22 October 2010 - 05:59 AM
What I saw is that there is still a pellet left in my sample lysates so the supernatant was not transferred to a fresh tube.
Can that have an effect?
I wonder now to change my lysis buffer. Is that perhaps an option?
I'm confused by this sentence. Could you explain in detail how are you preparing your sample?
Also, when looking at phosphorilated proteins is important to add Na3VO4 in the lysis buffer to prevent de-phosphorilation, have you added this to your buffers? I think RIPA buffer already has it in it, but not sure about others.
Posted 23 October 2010 - 07:53 AM
I cut a piece of my frozen tissue and homogenize it in my buffer.
Next I centrifuged them for 5min and stored them in -20°C. So I forgot to
put the supernatant in a fresh tube...
Then I performed a BCA-test, measured the protein concentration.
And when I use my samples for WB, I make a solution with MiliQ,the protein lysate and sample buffer.
Next I incubate them for 5min at 95°C.