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To clarify my experiment, the PBMCs were pretreated with anti-RAGE an hour prior to stimulation with AGE-BSA. The concentrations of anti-RAGE used were 0.1, 1 and 5ug/mL and there is a dose dependant increase in TNFa production when co-cultured with AGE-BSA after 48 hours. This had led to the question whether or not the anti-RAGE completely blocked RAGE. (Which part of the epitope is this antibody raised against? Is there an available full length anti-RAGE that blocks it and inactivates the AGE-RAGE pathway?) TNF-a was one of the many pro-inflammatory cytokines that was produced by the PBMCs upon stimulation of RAGE. This was chosen to determine the optimal stimulation with AGE-BSA. Consequently, we also used TNF-a to monitor the inhibition (in this case, activation) of RAGE with the anti-RAGE.
Asked on Sep 15 2011
Thank you for your patience. The lab send the follwoing information and suggestion: (1) The immunogen of this antibody is the extracellular domain (based on the UniProt database entry Q15109: residues 24 to 344). (2) The customer could determine whether this antibody is agonistic by measuring TNFa in cell culture media when ab89911 is added alone (without AGE-BSA). We'd suggest to try this. I am not sure if you have done this already, but maybe it would help to measure TNF-a under the following conditions: ab89911 alone AGE-BSA alone ab89911 + AGE-BSA together Titrate ab89911 and AGE-BSA separately as well as together. You might want to use similar concentrations as the lab did for their ELISA to make sure you see the blocking effect (2 μg/mL ab89911, 0.5 μg/mL of Biotinylated-AGE-BSA). How much RAGE do you think is expressed on the PBMCs? Should after trying these tips the results still not be satisfactorily, I'd be happy to replace, credit or refund the antibody. I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.
Answered on Sep 15 2011