Protocol Online logo
Top : New Forum Archives (2009-): : SDS-PAGE and Western Blotting

Lower MW bands than expected. - (Jul/08/2010 )

I'm a grad student looking at multiple proteins via western blot. The very specific (clean and one-band result) antibodies I have used have lower molecular weights than says on the datasheet from the companies. For example, the result I get from one antibody gives me a very specific band around ~38 kDa with whole cell lysates, where as the company expects you to find a band ~45 kDa whole cell lysate (NE). For a different specific antibody I have a ~50 kDa when the expected band should be around ~58.1 kDa.

I'm suspecting it could be a gel concentration problem...I use a Tris-HCL gel 4-20% from BioRad. Is the gel I'm using an issue/gel electropheresis issue or can it be the protein is undergoing post-translational modification/splicing during transcription level?

Thank you for those who can!



I think if it is consistent then it could be a gel issue, but I've hugely differentseen it often that the size is different in a different cell type, species. Also, if the abundance hugely different, it can appear slightly different in size,too.
HOwever, I would put my money on modifications.
Often, you see differeent sizes to expect with different antibodies for the same protein.