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Anti-Dab1 (phospho Y198) antibody (ab5673)

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1 question for ab5673

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Question 1

Wednesday 31-March-2004

I have a couple of questions about catalogue # AB5673, the rabbit antibody against Dab1 (phospho Y198). First of all, are you able to disclose the exact region of phosphopeptide that was used as immunogen?

The second question relates to the Western blotting protocol that was used with this antibody. On the data sheet, it is stated that "Lysates prepared from HEK293 cells transfected with wild type (1, 2) or Y198F mutant mouse Dab1 (3, 4) and left untreated (-) (1, 3) or treated with H2O2 (+) (2, 4)". My customer would like to know why there is a treatment with hydrogen peroxide for some of the cells. (He has looked up one of the references on the data sheet but saw no mention of H2O2 being used.

I look forward to hearing from you. Thank you for your help.

Best Regards.

ANSWER:

  

Thank you very much for your patience. It took some time to get some feedback about this product.

The immunogen is considered proprietary. The antiserum was produced against a chemically synthesized phosphopeptide derived from the region of human Dab1 that contains tyrosine 198. The sequence is conserved in mouse, rat and chicken. You can tell the customer that the sequence is usually 5-10 aa long generally centered on the phosphorylated residue. So if they know the sequence 5 aa on either site of this then that would encompass the immunogen. That is as much as we can share.

H2O2 is an inducer of apoptosis/stress pathway. So in lane 2 it induced Dab1 to phosphorylate at tyr 198 and thus the antibody specific for that can bind to it and you see the band. In lane 4 is the mutant that cannot be phosphorylated at that site, even with stimulation from H2O2 so there is no band.

I hope this will help.

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