21 replies to this topic

[mdfenko]

without sample (loading) buffer for checking the native protein.

Edited by mdfenko, 20 October 2010 - 06:38 AM.

[rkay447]

HomeBrew, on 18 October 2010 - 11:49 AM, said:

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).

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?

[Blackeyed]

I`m also considering that the sample preparation may be the problem.
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?

[rkay447]

RIPA is a great lysis buffer for western blots (not the best for interactions studies though).  This is the buffer I prefer to use when harvesting cells for western blots.  Again, I highly recommend you ponceau the blot after transfer just to see the separation and transfer went well.

[Blackeyed]

So guys, I tried the dot-blot. I even did two.
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?

[almost a doctor]

Blackeyed, on 18 October 2010 - 05:42 AM, said:

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.

[Blackeyed]

Well; what I did: I made a buffer with NaCl, Tris, EDTA, Glycerine and Halt protease inhibitors.
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.