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Western blot fail, time and time again - (Oct/01/2010 )

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Blackeyed on Mon Oct 18 13:42:13 2010 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.

-almost a doctor-

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 -20C. 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 95C.

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