Hi. What represent clone 1 and 2? ...

If you centrifuge the cells after lysing in RIPA you might lose histones in the centrifugation pellet , and depending on the cell treatment. The total protein profiles ,per se, look different in lanes 2 and 3. It is not good idea to lyse in cold if you study histones. If you explain those details to me, I might help further .Regards

Hi Cristsian,

Thank you for the comments!

The clones are single cell clones of stable KDM3B knock downs. No treatments here, just baselines.

I will say I've used RIPA and lysed on ice in the past for histone marks (H3R8me, H4R3me) without any issues. I would also note that these samples were prepared in parallel and lane 1 and lane 4 have strong histone signals. I'm going to try a DNA digest next to see if that helps with histone recovery though I'm still puzzled why these samples would behave so differently when prepared so similarly. Please let me know if there are any other experimental details I can provide.

Thanks,

Fiona

AS your ponceau stain suggest , your samples 2-3 has undergone proteolysis (see the smears and the low amount of HMW proteins) so the signal of H3methyl and H3 are booth consistent. I would suggest, you sonicated slightly your samples..your band will be sharper

Thanks for the comment! Would this be from something other than enzymatic digestion? I did add protease inhibitor and all the samples used the same lysis buffer at the same time.

yes , this is weird . if your sample do not need to be in a native lysis buffer (ie: for use in IP expt) . try lysis in a fully denaturing buffer 1%SDS (with out detergent, and sonicate)

. and quantitated with ...Does SDS affect the BCA assay?

Note: 5% SDS is compatible with the BCA Protein Assay.