I use agarose gel electrophoresis to study enzymes for which I can stain for enzyme activity, but I want to identify the enzymes by specific antibodies on a Western blot. Would "Southern" blotting work? I use PVDF membranes.
Mary,
Our lab runs high molecular weight glycoproteins on horizontal SDS-Agarose (2% agarose) gels. For this we run the gel, transfer to a PVDF membrane using the capillary method and then proceed with a pretty standard western blotting process of blocking followed by primary antibody washing secondary antibody ECL development. I hope that this helps for reference, here is a citation of one of the paper in which our lab has used this method. As far as southern blotting is concerned I don't think this is the answer as this involves probing with specific, labeled oligonucleotide sequences whereas the method I have described will still use antibodies.
NCOA3-mediated upregulation of mucin expression via transcriptional and post-translational changes during the development of pancreatic cancer S Kumar1, S Das1, S Rachagani1, S Kaur1, S Joshi1, SL Johansson2, MP Ponnusamy1, M Jain1,3 and SK Batra1,3,4
Thanks for replying.. I only mention "southern" blotting for the description of setting up the"capillary' method. I am testing for enzyme activity in a non-denaturing, non-sieving system but want to confirm the enzyme's identity by an antibody. Th enzyme may be a multimer, but is not glycosylated. You did not give the exact reference to your article. I would like to read it.
I have done exactly this with intact phage virions, which migrate in agarose thanks to the enormous amount of nucleic acid they encapsulate.
I used wet transfer to nitrocellulose just as with SDS-PAGE, albeit adapted to the thicker form of the agarose gel. I didn't change the buffers at all - 0.5x TAE for the gel and 20% methanol in Tris-glycine transfer buffer, 25 mA overnight at 4 °C. The next day I blocked & probed as usual to fine results.
Here is the full citation I hope that it helps.
S Kumar, S Das, S Rachagani, S Kaur, S Joshi, SL Johansson, MP Ponnusamy, M Jain and SK Batra. NCOA3-mediated upregulation of mucin expression via transcriptional and post-translational changes during the development of pancreatic cancer. Oncogene (2014). doi:10.1038/onc.2014.409
Also the use of SDS for you in this case would be advised as your enzyme will still need to be coated with a negative charge in order to run through the gel properly. I hope that this is helpful.
Andrew
Thanks for replying. I am abandoning electroblotting.
My problem has been solved by a colleague who suggested that I try the classic "Southern" method.! I was surprised that it worked so well. Of course I used the TRis-glycine buffer whch we call "Davis" since he devised it. It is what Laemmlli modified for SDS-=PAGE by adding SDS..
I was ready to abandon agarose until I came across the VAGE method of Warren et al. Electrophoresis 2003 24: 1695 which I am about to try to see my 2 MDa mussel protein on a blot.
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