Hello all,
We are interesting in studying p-H2AX in lymphocytes. At the same time we see some other proteins, the bottom of the membrane is cut and incubated for p-H2AX (cell signalling, 1:1000).
The problem? I put an image (the membrane is cut at 26 kd): We see like four bands under.
Lysates are prepared after 30 mins extraction with RIPA buffer with protease/phosphatase inhibitors (as we have seen some papers studying histones where they use RIPA, and an acid extraction is not convinient for the other proteins we study). Gels used are at 9%, but the front didnt scape and we could see the mark of 15kd of the visible protein marker. Transference is done for 1 hour at 100v in cold transference buffer. Primary antibody and secondary antibody are prepared in Phosphoblocker-TBST.
Moreover, these bands could be seen after 10 mins of exposition (maybe, a lot, although we don't expect a high activation of the DDR in basal conditions). Does anyone know if, without activation, these bands can be seen? Would you think we could see a single, beautiful band irradiating cells (UV damaged) in these conditions?
Hi Robert, I am not familiar with your assay, but why do you cut the membrane?
If it is because other bands are positive, then I am afraid that you are using a bad antibody.
Plus, if you are using an HRP secondary and chemiluminescence to detect your protein, then a 10 min exposure is far too long.
Do you have a blot of your cells that you only incubate with the secondary Ab and expose for 10 min as a control?
Thanks for the answer. We cut the membrane because we'd rather blot each portion of membrane separately rather than blotting and reblotting and reblotting...About the method, we are using chemoluminiscence (ECL). We did controls with just the secondary and these bands didn't appear.
We think it is something about the protein or the antibody.
Hi Robert, I don't understand your rationale for probing sections of the membrane and I don't understand what you mean by "blotting and reblotting".
Anyway, to me, 10 min exposures using ECL indicates that you have very low protein concentrations in your lysates or you are using a bad primary antibody.
Can you do IP of your lysates to increase the concentrations for the gel?
If your antibody is specific to the phosphorylated version of your protein, is it possible to add dephosphorylated fractions as a control? These lanes should have no (or atleast greatly reduced) interaction with your protein of interest and could be used to help you rule out non-specific bands on your gel. Also, if you want to see if your primary is working properly, I would think you need to blot the whole gel. For the blot and reblot issue, are you looking for multiple proteins so you would have to strip and reprobe?
Yes, we are studying at the same membrane proteins which weight 36 and 55 kd
Hallo,
can you please tell me if you still remember. what were these 4 bands. because i have now the same problem yours. with 4 bands.
thanks
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