Anti-RBP4 抗体 [EP3657] (ab109193)

I am happy to hear you have already reduced, denatured, and protected your samples from proteases. The only other attempt that I think is worth your time is running a WB with mouse serum and kidney lysates, especially since the wildtype levels of Rbp4 (when 30ug of whole brain cell lysates were loaded) barely produced a detectable WB band in the paper you referenced. If the antibody detects Rbp4 in the positive controls, then you know the antibody is properly detecting Rbp4 and that the levels in your sample may be very low, necessitating a much higher sample loading amount.

I look forward to your reply and hearing whether or not the positive controls yield the expected Rbp4 band.

Thank you for contacting Abcam and providing your protocol information.

I am sorry to hear that you have been experiencing issues when using this antibody. This product is covered by our Abpromise guarantee. According to Abpromise guarantee, if this product has been purchased within the last six months, we are happy to provide scientific support and to replace or refund the product should we not be able to help.

Upon reviewing your protocol, I have a few additional questions/troubleshooting tips:
1. Did you reduce and denature your samples? We recommend adding Beta-mercaptoethanol or DTT along with SDS and boiling with your sample 95-100oC for 5 minutes.
2. Did you add protease and phosphatase inhibitors to your lysis buffer?
3. Did you run a positive control? If you have access to human plasma, HeLa or HepG2 cells, or liver or kidney tissue these have all been used as positive controls for RBP4. While the mouse atlas may site specific examples of RBP4 expression in mouse brain, the actual RBP4 protein may be present at very low levels or not translated into actual protein. Gene expression does not guarantee translation into the endogenous RBP4 protein. A positive control will help determine if the antibody is properly detecting RBP4 or if the protein is not present in detectable levels in your sample.

These will not only help me to fully understand the problem but are essential should further testing of this product be needed as well. Could you also provide the Abcam order confirmation number or the PO and date of purchase for this product?

Thank you for contacting us.
ab109193 has not been tested for neutralisation and we have no empirical data to support the application at the moment.
I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.
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