I was wondering if anyone has ever noticed anomalous migration during PAGE
of proteins tagged with the FLAG epitope.
We used a monoclonal antibody to screen a library and obtain a cDNA for
the corresponding antigen. In Western blots, the monoclonal detects a 69
kD band in normal rat liver extract, and so does an antipeptide antibody
raised to the protein predicted from the cloned cDNA. However, we put the
cDNA in an expression vector so the protein is expressed with the FLAG
epitope fused to it, and when we run the Western the band is at 97 kD, as
detected by anti-FLAG, the monoclonal and the anti-peptide abs. I am at a
loss to explain the size difference. According to sequence data, the
start and stop codons are ok, and the size predictions by computer
analysis say 65 kd, a reasonable match to the 69 kD we see in the native
protein.
We are in the process of trying to cleave off the FLAG prior to running
the gel, and we're also putting the protein in a different expression
system, but I was curious to find out whether anyone else has ever had a
similar experience, or can offer any suggestions as to what might be
happening.
Thanks.
--
Deborah Britt, Ph.D.
Department of Medical Oncology
Rhode Island Hospital
