The molecular weight that the bands should show up is at 150 kDa, however a western blot of several cell lines reveal the band at 37kDa uniformly. Could there be any reason why? My literature review and BLAST searches for the sequence binding only reveals proteins/peptides over a 100kDa.
It seems that your antibody is non-specifically binding with some other abundant proteins (probably, beta-actin). This could be due to incomplete blocking of your membrane or antibody is not good. To verify this,
1. After transfer, do a Ponceau S staining of the PVDF membrane and take the picture. Here beta-actin will be the most prominent band present around 37kDa-42kDa.
2. De-stain the PVDF membrane and proceed to immunoblot.
3. Take the image and check whether the band you are obtaining is overlapping with beta-actin band in Poceau S stained image.
4. If yes, your blocking is inadequate or antibody is non-specific.
Use 5% BSA to block 1hr at room temperature with gentle rocking or perform overnight blocking at 4 degree Celsius.
Regards,
Siva
Hi,
membrane proteins are difficult to analyze. Make sure to choose an appropriate protein isolation protocol for membrane proteins and a low percentage for your SDS-PAGE. Otherwise you will not be able to detect the receptor protein. The large proteins are blotted with a very low efficiency. It is a good idea to use a total protein labeling (https://www.dyeagnostics.com/site/en/products/spl/) to monitor your blotting efficiency to see if you get enough target onto your blot.
Good antibodies for receptors like VEGFR are not easy to find. I have never made good experiences with Santa Cruz to be honest. I always preferred Cell Signaling antibodies. To be on the safe side it is a good idea to verify your target via mass spectrometry.
I hope I could help.
Best,
Kristin
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