Hej,I'm trying to Western blot with histone samples (purified by acid extraction), but I keep getting very diffuse and ugly bands. In higher MW, the (aspecific) bands look nice and clean.
I have tried both wet and semi-dry blotting, I also loaded total cell lysate in parallel, and old histone samples which have worked before, but which also look ugly in the
Hello , I did the same work as you do . You can try to Run 10 or 12% SDS-PAGE. what i do is wet bloting. (condition 80v for 2.5hrs) i hope it can help you.
Hi, in my opinion, to resolve the low molecular weight proteins you need higher % of SDS-PAGE gel to prevent the protein bands from diffusing. Giving the gel enough time to strengthen prior to using it has some effect on the protein bands migration. Running the gel at low voltage also helps with keeping the protein bands crispy.
I'm sorry I havent specified enough. I have tried both an 18% gel and an 4-15% gradient. We buy the gels precast.
please check the following link:
http://www.abcam.com/protocols/western-blot-troubleshooting-tips
15% gel works for histones. Also, load the protein sample depending on the abundance of histone of your interest.
I solved the issue by changing to BIS-TRIS gels. Looks textbook like!
e.g. as submitted here: http://pabmabs.com/wordpress/?p=4659
Hi Veerle! I need to do WB for histones in brain samples. Did you extract the nuclear fraction of your samples? Did you use HDAC inhibitors?
Thanks
Angela Nicola
I extracted histones by acid extraction (several protocols online) - I needed a pure fraction for mass spectrometry later on. It is not a very difficult protocol, and something I would recommend if specifically interested in histones and histone PTMs. I have compared cell lysate with histone fraction to test antibody specificity, and did not see the same bands. In the histone fraction, I saw 1 single band of correct MW, but the same band was very weak in cell lysate, while having many more aspecific bands. See here for example of histone H4: http://pabmabs.com/wordpress/wp-content/uploads/2017/05/h4-ab7311.png
I had added protease inhibitors, HDAC inhibitors and phosphatase inhibitors since we planned on profiling the global PTM landscape. Depending on your research question, you need to add more or less inhibitors. Generally, I would advice to add them; several enzymes get on the loose when lysing the cells first, likely changing the PTM profile.
Good luck!
Depending on the composition of your Lysis buffer, some proteins arent fully soluble. You should try a SDS containing buffer for tissue lysis. Or if samples are already in RIPA buffer or similar, add Laemmli buffer and pass samples through a 25G needle.
The following paper by kevin janes in demonstrates that some proteins are not totally soluble in RIPA/NP40 buffers (fig1).
Article An Analysis of Critical Factors for Quantitative Immunoblotting
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