Using Bcl2 antibody from Santacruz(Sc-509), I am observing bands b/w 15 and 10 kDa, instead of expected around 26. How should I proceed in such a case?
We are using a mouse cell line for our experiments.
Additionally, I am attaching the image of the developed Bcl2 expression.
Have you checked the antibody with human cell lines? Does it give a band at expected size? Sometimes the antibody may recognize an internal translation or proteolytic product of Bcl-2 and give the 18-15 kDa lower band. Check with human cell lines (positive controls).
Hey Vray, Hope everything is fine?
The lower band is may be due to proteolytic cleavage or your protein undergoes some post-translational modification like phosphorylation or glycosylation, which ultimately results in differential mobilities on SDS-PAGE. Also, look for the specificity of your antibody, is it detecting another protein too??
Are you using protease inhibitor during your preparation?
Also, Santa-cruz provides a broad range for Bcl2 antibodies, which one are you using?
Is it possible to cut the band and get the mass spec done? or affinity purify the band and get the N-terminal sequencing done?
Bawa, I am fine, How are you?
The antibody detects for Bcl2 with its epitope between aminoacids 41-54. I did blast analysis for this sequence. All the homologus proteins were of much higher molecular wt. No we are not using proteolytic interventions or inhibitors. At this point getting mass spec or sequencing may not be the immediate proirity. May be I shall try to compare my cell line with a human cell line for Bcl2 as recommended by Debanjan or for post translational modifications at you suggested. Further ahead I shall proceed with mass spec or sequencing.
Thanks a lot for your response
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