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Thank you for your reply and suggestions. Since your last e-mail I have spent some time optimizing western blot conditions using the SEMA4A (ab70178) antibody in a range of cells lines. I am still seeing bands of higher molecular weight (~120 and ~130kDa) than predicted (84kDa) but they are consistent. Also we bought a SEMA4A overexpression lysate in which the antibody detects a band at ~84kDa-so I am certain the antibody detects SEMA4A. I have one final query, on the spec sheet the antibody is raised against a peptide from the internal region of SEMA4A. Could it be cross-reacting with SEMA4D? I ask because SEMA4A and SEMA4D are highly homologous and I wonder if there might be cross reactivity, as SEMA4D has a Mw ~127kDa. Any information you can send me on this matter would be very much appreciated.
Asked on Nov 29 2012
Thank you for your email. I have more information regarding the immunogen: The antibody binds to the PSI domain. Alignment of our immunogen sequence with human SEMA4D ACC# http://www.uniprot.org/uniprot/Q92854 shows a 25% identity in an 8 residue overlap. It is unlikely that the band at the higher molecular weight is SEMA4D as the homology is low. It is more likely to be the glycosylated protein. I hope this information is helpful. Just let me know if you have further questions.
Answered on Nov 29 2012