Our lab purchased a antibody from Abcam, and on the product link it shows that the protein size and the detect size are both around 90. However when I run my own sample on the gel and detect with this antibody, my band size is only about 75. There is no other band on the blot and the background is pretty clean.
Another thing I should mention is that I have non treated sample and overexpressed sample. I do see the band intensity increase for the OE sample. So the antibody should be detecting the right protein. But not at the correct size.
Does someone know why this is happening? Is it because of the % of the gel I used? Or because the protein is somehow degrade?
Thank you!
This looks like the protein you are looking for. To be sure use 8% PAG, load 3 times less marker that you loaded on the gel in the picture, and reduce current in electrophoresis to get "nicer" bands. Also I would load TCL from another cell line as a control because protein can be differently sized in different cell lines.
Hi Jianing
It is possible that your sample contains an isoform of the protein you are looking for. Usually Abcam gives precision regarding the type of sample they are using (cell lysate, which cell line, mouse sample, recombinant protein etc...). Maybe abcam is showing a precursor of your protein, hence the higher apparent MW. Since you have only one band this should your protein. I would recommend that you check existing isoforms of your protein and their specific expression using a protein database such as Uniprot. You can also check if you protein is supposed to have glycosylations or not.
Good luck
Pierre-Marie Andrault Thanks for your suggestion. I looked it up on Uniprot, it shows this protein has 1 described isoform(796aa) and 4 potential isoforms(40-50aa). So what I'm detecting is probably the long isoform. My antibody detects the very end of the C terminus, so if it is truncated, it would be in the N terminus. I can try to see if there another antibody target the N terminus.
Could you give me some thought on when should I decide if it is ok for me to proceed if another antibody is detecting at 75 as well?
Thanks,
Winny
Mateo Glumac Thanks for the suggestion. I'll make the adjustment and add a few controls from different cell lines.
Mateo Glumac I just fond out this image from another supplier website for an antibody against the same protein. They tested all type of cell lines and I used HEK293T. Should I be worried that somehow my blot was detecting the lower faint band showing on this blot?
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