I transfected 293T cells with a flag tagged protein (40kDa). After performing the westernblot and Flag antibody treatment I was supposed to see a band on 41 kDa. Instead,that I see clear band on 20 kDa. I am curious to know why I am facing this issue. If anyone could clarify, that would be a great help. Thanks in advance.
Hi there,
Is the Flag at the Nter? If so there are 2 obvious options = the FL protein gets proteolyzed or there is a stop codon in the middle of the ORF. If Flag at Cter only degradation can explain... Do you add protease inhibitor when lysing the cells?
Thanks, Dr. Dominique Liger for your answer. I inserted the Flag tag in the N-terminus of the protein sequence. Yes, I added both protease and phosphatase inhibitors while lysing the cells.
Hi again,
If you have the possibility to perform lysis in denaturating condition (in SDS and urea containing buffer for instance) to limit protease activity... To rule out the fact that lysis in native condition may trigger the degradation.
- Badges
- Science topic
- Similar topics
- Biological Science
- Biochemistry
- Biomolecules
- Nucleic Acids
- RNA
- Small RNA
- microRNA