Dear Zynep

Please see an article

Cancer Biol Ther. Sep 15, 2011; 12(6): 484–493.

Published online Sep 15, 2011. doi: 10.4161/cbt.12.6.15956

PMCID: PMC3218590

The DNA damage mark pH2AX differentiates the cytotoxic effects of small molecule HDAC inhibitors in ovarian cancer cells

Andrew J Wilson, et al

See Figure 5

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3218590/figure/F5/

Looks like the double bands may be an artifact of gel separation.

Which antibody are you using for the p-H2AX? I wonder if it is detecting both the phospho and total H2AX forms. In response to UV, we don't see changes in total H2AX protein.

Here is a recent publication from our lab. The antibodies we used are mentioned the methods section. Hope it helps.

http://www.ncbi.nlm.nih.gov/pubmed/24730569

Thank you for trying to help.

I'm using the one from Cell Signaling.

In your publication you only show y-H2AX, and it's not stated whether it's phospho or total.

In the supplemental figures, we have both the phospho and total H2AX. The y-H2AX (S139) antibody is from Millipore. The total H2AX (C-20) antibody is from Santa Cruz. You can find the supplemental figures online.

Maybe my question was not clear:

What can one do, if there are 2 bands instead of one, and one is sure the ab is specific? Whether it's artifact or whatever you want to call it..

I would recommend that you run a positive lysate control for y-H2AX staining and then see what band(s) come up in the positive control. Using the lysate of another cell type/line might help you to determine which band is the correct one.

It could be the mono-ubiquitinated form of H2AX.

Can you estimate the apparent molecular weight of the upper band ?

It's above 15kDa (the last blue band of PageRuler Plus Prestained Protein Ladder). I attached the picture with the drawn band.

Well it's quite difficult to evaluate the size of the upper band with a single marker.

It should migrate near a ~25kDa size marker to fit with H2AX-ub.

Then it's not, because the 25kDa is far.

Could it be that it's detecting another member of the H2A family?

like here on CST page http://media.cellsignal.com/products/images/171183/294084/2577_wb_jb_040105.jpg

However, my colleague is using the same antibody, and he never got double bands but once.

Assuming you have a good overlap between the H2AX and gH2AX (i.e. H2AX signal is present in both "unmodified" and "modified"and therefore you are looking at the right (H2AX) population; hard to tell since the gels are reprobes), looks like the upper 17 kDa band might have additional modifications (assuming your 15 kDa marker is accurate). If you're not sure where H2AX should normally migrate on your gel system, just ponceau stain: most histones (except H4) should pack up at the bottom and be clearly visible as a single band around 15 k...would tell you where to expect to find most of your H2AX.

Agree with other comments that your total H2AX fluctuates A LOT (over 6 h treatment...)! Very unlikely to represent faithfully the whole H2AX population. Which brings us to the main point: have you considered the possibility that your secondaries cross-react (donkey anti-goat with goat anti-mouse for instance). Would explain why you pick up only gH2AX (dominant signal) but doesn't explain why you have 2 bands. The 17kDa likely represents additional modifications...or a crossreactivity with another phosphoprotein.

Hopes this helps

Hi there,

one thing at first, I'm not sure if sb answered you on this, gamma H2A.X is nickname for the phospho S139 H2A.X modified histone ;), It took me also a while to understand that.

Secondly, we checked the total H2A.X prot. level within different treatment conditions on ICC a the intensity was rather stable.

Did you check also some hcg like beta-actin or GAPDH, if their levels correlate with the total H2A.X, it might be, just problem with the sampling on gel. Even you have the same prot. levels/well, might be, that DNA, if not completely shared, causes the sample higher viscosity and you might load different volumes into wells.

As for your detection by WB, Cell signalling are really good Abs, so to think, that there would be the problem is probably the last to think of.

Since the double bands are so close to each other and they show similar intensity profile, it is possible, that when you developed your films, that you moved it on the membrane and as you had probably strong signal coming from the bands, it showed "shadows" looking as real signals, that's one probable explanation.

Did you try to consider this?

The last thing, we used in lab the Abcam Abs, rabbit mAb against gammaH2A.X and rabbit polyclonal against the total one and it worked perfectly.

Hopefully sth from the above helped you.

Hello again!

Thank you all for your valuable contributions.

None of the above mentioned points were the case, so here's what I did:

I re-isolated proteins and used a different protocol this time: instead of 30' lysis buffer followed by ultra-sonification, I froze my extracts (in lysis buffer) in liquid nitrogen and after defreezing immediately centrifuged them at high speed.

Afterwards I used the usual protocol for WB.

Now I have single bands for yH2AX. Since my colleague never had double bands, I think the ultra-sonification could be bad on some proteins depending on the cell line - so that some are more sensitive to ultra-sonification (in my case: SW480) than others (DLD-1) (? correct  me if that's wrong).

I hope that will be helpful to others experiencing the same problem.

Well you may be right indeed.

We also experienced cleavage of some proteins when lysates were sonicated in the presence of SDS (not with other non ionic detergents).

But note that in this case the extra bands migrated with lower sizes.

It is still intriguing that you observed an extra upper band.

Anyway it's OK if you finally solved your problem from a practical point of view, even if not from a theoretical one.

Hello :),

I'm happy that you solved your problem.

And agree with what you said about the

cellular differences you encountered.

...Never follow the protocols without thinking

about them,  when it works on the first time,

you might be just lucky ;)... Each system is different

May I know how long have you frozen your extracts in the liquid nitrogen? Thanks. 

Very shortly - until the eppis were frozen and the clicking/knack noises were over.

Btw: I defrost them on ice.

Sorry this is an old thread--when you are transferring to detect gH2AX, how long do you transfer for? I'm having a hard time detecting using the Cell Signaling ab, and wondering if I'm transferring for too long (60 min). Thanks for your help!