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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 contacting us. I have passed on your experimental details to the lab for more input, and will let you know what they suggest. Regarding your questions: 1) Which part of the epitope is this antibody raised against? The antibody was raised against the extracellular domain which is about 300 residues long. The exact epitope (which is usually 5-10 residues) has however not been mapped. 2) Is there an available full length anti-RAGE that blocks it and inactivates the AGE-RAGE pathway? I am not sure if I understand this question correctly. AGE would be binding to the extracellular domain only. Thus an antibody against full length RAGE would not make much difference or even block binding. We do not carry any other antibody that has been tested so far in inhibition studies - only ab89911. Lets wait if the lab has any further suggestions and we can discuss further how to proceed. I will be in touch. In the meantime, please do not hesitate to contact us if you need any more advice or information.
Answered on Sep 15 2011