I am doing WB with HO-1 antibody (from Enzo) on HepaRG cell lines and all 3 times I received the only band around 60-70 kDa while HO-1's MW is around 31 kDA. The background is completely clean. Hence I assume that this could be the dimer of HO-1.
What should I do to prevent the dimer or how could I confirm that was the dimer of HO-1/
Thank you
Did you perform native or reducing PAGE before the WB application? In Leammli buffer with heat treatment, as strong reducing conditions, it must be converted to its monomer if the observed protein is a dimer. Alternatively, using WB, you may fractionate lysate through SEC and verify the fraction of interest at 30 and 60 kDa. Circular dichroism (CD) spectroscopy and MS in some cases may also be beneficial to clarify oligomerization. Native PAGE fractionation and band excision followed by bottom-up proteomic identification can also report dimerization.
Good luck...
If the protein is a dimer, you would see two bands on the Western blot, one for the monomer and one for the dimer. If the protein is a monomer, you would only see one band.
- Badges
- Science topic
- Similar topics
- Biological Science
- Immunology
- Antibodies