Anti-MELK antibody (ab9696)

BATCH NUMBER 158815 ORDER NUMBER 41882570

DESCRIPTION OF THE PROBLEM I have two bands in Jurkat extracts, one around 120K, and another around 65K. The commercial GST-MELK protein migrates around 100K. I read the explainations, however it seems to me that the MS was done on the commercial protein, not the 120K band. Is this correct? I would be much happier to spend my money and time if the 120K band was identified by MS.

ANTIBODY STORAGE CONDITIONS 4C

ANSWER:

I would like to reassure you that the 120-150kda band we consistently detect in western blots differing from the predicted size of 72kDa. Complete blocking by the immunising peptide is observed with peptide neutralisation experiments. It is to be noted that MELK is a member of AMPK family, which has several consensus glycosylation sites. Glycosylations are known to affect migration of proteins on a SDS-PAGE [Hames, B.D and Rickwood, D., eds. Gel electrophoresis of proteins: a practical approach. Oxford University Press]. Further the activity of this antibody was confirmed against recombinant, GST fused MELK protein by Western blotting.

I have added this information to the datasheet and hope this will help you,

The predicted size is listed to be 120-150kd on the datasheet, when other literature predicts 70kd.

ANSWER:

Thank you for your enquiry.

I found further information regarding your question. It appears that the protein may be glycosylated, leading to slower migration in the gel.

During testing, the band that was consistently detected in western blots differed from the predicted size of 72kDa. Complete blocking by the immunising peptide is observed with peptide neutralisation experiments. It is to be noted that MELK is a member of AMPK family, which has several consensus glycosylation sites. Glycosylations are known to affect migration of proteins on a SDS-PAGE. The activity of this antibody was further confirmed against recombinant, GST fused MELK protein by Western blotting.

I hope this information helps, please do not hesitate to contact us if you need any more advice or information.

We have been studing MLEK function in CNS for several years, and are now thinking of purchasing MELK antibody from your company. I am wondering MELK product should be about 70kD but in your catalog, it is written as 120-150kD. Could you tell me the exact size of this product? And we would also like to know if your antibody works for ICC. Thank you for your cooperation.

ANSWER:

We consistently saw a band at 120-150kDa by Western blot in both Jurkat and HeLa cell lines. However, we don`t have an explanation for this discrepancy, given the predicted molecular weight of approx 70kDa. We would like to inform you that one of our customers who has purchased this antibody contacted us recently. The specificity of this antibody was confirmed in his lab against recombinant MELK (KIAA0175) protein on a Western blot. He also had the reactive band excised and IDd by MS further assurance that the recognized protein was indeed MELK (KIAA0175). What size does your recombinant protein run on SDS-PAGE? Do you have any evidence of alternative splicing of MELK? Have you used our antibody on endogenous protein samples and if so what did you observe? We would welcome any feedback in this respect from researchers who have tried using this product.