I am trying to detect changes in Histone PTMs with or without treatment in nuclear extracts from cell lysates.
I use a Hypotonic buffer (20 mM Tris pH 7.4, 10mM NaCl, 1mM MgCl2, 0.5mM EDTA) supplemented with protease inhibitors in order to obtain a soluble cytoplasmic fraction. I then use a hypertonic buffer (20mM HEPES pH 7.9, 420mM KCl, 1.5mM MgCl2, 0.5mM EDTA) supplemented with protease inhibitors on the nuclear pellet in order to obtain a soluble nuclear fraction.
Using Western Blotting, I've tried detecting Histone PTMs using two antibodies: Anti-Acetyl H3 (EMD Millipore, Cat no# 06-599, 1:2000) and H3K9ac (Abcam, Cat no# ab4441, 1:2000) and I get very strong bands at ~72 KDa and not at the predicted MW (17kDa). When I incubate with Total H3 (Abcam, Cat no# ab1791), I get bands at the correct MW (17kDa).
I also tried fractioning out the chromatin, running it on a WB and still get the same results.
Has anyone else ran into this issue and/or know how I can correct it?
Thank you!
Hi there,
Just in case, I am wondering that the cell line you're targetting:
1. Does it have any published data for the same cell line?
2. Is this specific antibody is working alright with your cell line?
These are very basic points to be accurate in the said experiment!
Do you run that positive control on the same gel and get a band? Like there is no possibility you are running your low mw bands off the gel. Also, no expert, but seems your heavy band is spot on as a tetramer, would you expect a reducing gel to be enough to cleave all di, tri, etc. mers? Good luck!
Dear Rebecca,
there are several pitfalls in obtaining a concentrated fraction of nuclear proteins. First of all, you need to hit the right amount of cells in the right volume of buffer to effectively destroy the nuclear envelope. Have you checked the process under the microscope? In most cases, you will need some vortexing and/or NP40 to achieve this. You want to try another protocol?
Regards
Boris
Thank you Boris Schmitz for your recommendations! I ended up trying another protocol and it worked!
Best,
Rebecca
Pratibha Srivastava thank you for your recommendations. We have done antibody testing on a control cell line and it worked. Data on this has not been published for my cell line (mine would be the first) but I found a protocol that worked! Thank you again for all of your help!
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