I am trying to use western blot to show the p65 translocation from the cytosol into the nucleus upon LPS induction in Jurkat, BJAB and macrophages. But I'm detecting Beta-Tubulin in my nuclear extracts. I don't know if it is normal or if there is something wrong with my nuclear extraction? And if it is normal, which other protein(s) can I check for to make sure my nuclear extract is pure?
I don't get any TBP in my cytosolic extraction.
I use swell with low-salt buffer/dounce/high-salt buffer/dounce method to do the nuclear extraction.
I use alpha-tubulin. works well for me. You can also use, in most cases, GAPDH.
There some tubulin-isoforms in the nucleus that can be recognized by anti-tubulin antibodies. I had the same problem and now I'm using an antibody against a nucleolar protein.
histone H3 as a marker for nuclear protein as well. It is more an internal nuclear protein as opposed to a structural nuclear protein like Lamin A. I usually use both.
Lots of good suggestions. My 2 I use which always work are HPRT to mark the cytoplasm and H3 for nucleus. Good luck.
Be careful with GAPDH. An isoform of GAPDH is known as uracil DNA glycosylase and therefore may be "nuclear" protein under some conditions, including stimulation of B cells.
Sirover MA. Induction of the DNA repair enzyme uracil-DNA glycosylase in stimulated human lymphocytes. Cancer Res. 1979 Jun;39(6 Pt 1):2090-5.
H3 is very good nuclear marker, in similar experiment we used tubulin or actine for cytoplasmic fraction and H3 for nuclear fraction.
I used antibodies against the nuclear membrane structural protein Lamin to determine the nuclear fraction.
What about beta-actin. one paper from "Swiss Medical Weekly, the european journal of medical sciences, Swiss Med Wkly. 2011, 141. Shou Li and Et. al. showed p-NF-kB and beta-actin as a normalizing control. is this correct way? I attached paper. Please give me some idea if this is acceptable. Thank you.
If you have a look at Abcam website, they suggest: LaminB, CDK4, HDAC1, PCNA and a couple others... I've used LamniB before.
http://www.abcam.com/primary-antibodies/loading-control-guide
Tubulin should work, as it was said beta actin is another option, I usually do it with GADPH, any of them should work. If you are detecting tubulin in your nuclear extracts maybe you could try to wash twice or three times the pellet before to do nuclear lysis, I think it is more difficult to avoid nuclear contamination in the cytosolic fraction than the opposite so, you are doing it well, good luck
I use Gapdh and Lamin B for cyto ang d nuclear extracts. I am really interested in anyone commenting on Beta-actin though? can beta actin be found both in cytoplasmic and nuclear extract?
Beta-tubulin may be best for checking against cytoplasmic contamination of the nuclear protein fraction, although be aware that certain cancer cell lines have been shown to have nuclear beta-tubulin: https://www.ncbi.nlm.nih.gov/pubmed/12037579. Regarding beta-actin, we have found plenty of it in our nuclear extracts, as in https://www.ncbi.nlm.nih.gov/pubmed/16476775 , and GAPDH has been shown to migrate to the nucleus under stress conditions: https://www.ncbi.nlm.nih.gov/pubmed/20601085.
Good luck!
Hi Moshe; thanks a lot for great points and references. Actually I have personally experiences the translocations and/or contaminations mentioned in the references that you kindly shared. For a specific intention I am trying to find a protein with least possible shuttling.
Best Best Wishes
Beware of using b-actin since this will be in the cytoplasm as well! I would reprobe the membrane for cytosolic proteins (GAPDH, b-actin) after a nuclear membrane to check for contamination as indication of how efficient the separation of nuclear and cytoplasmic proteins was.
I use Histone H3 and beta actin as nuclear and cytoplasmic marker and they work well
I use SP1 for nuclear and BID for cytoplasmic fractions. B-actin seems to be present in my both nuclear and cytoplasmic samples in my case.
I uses SP1 for Nuclear fraction, GAPDH for cytoplasmic fraction, I saw B-actin present in both extracts.