For the best experience on the Abcam website please upgrade to a modern browser such as Google Chrome
We tried blocking with goat serum and that did reduce the background staining, but we are still unable to see any specific binding. Do you think the problem lies in our protocol or the antibody? Any feedback will be welcomed. I have attached a copy of our protocol. We were also going to try and incubate our primary antibody for 2 hours at room temperature instead of overnight at 4 degrees celcius. Let me know which you think is better.
Asked on Sep 05 2012
Thank you for your email.
I am happy to hear that you have been able to reduce the background. It is unfortunate, that the specific staining is not visible.
I am happy to help to troubleshoot, however it is difficult for me with the information I have. I have here some points, which might be worth considering - mainly in regards to the staining of the primary antibody - please let me know what you think:
1.) Are you able to detect the target proteins with these or other primary antibodies with this or another detection system?
2.) Do you know whether the TLR2 and TLR4 are present in the samples used?
3.) Have you tried another antigen retrieval method - such as for example heat induced antigen retrieval with EDTA buffer pH 9? Changing the antigen retrieval buffer can change significantly the results. Please see also the publication byEmoto et al., J Histochem Cytochem. 2005 Nov;53(11):1311-21.
4.) In regards to the background - have you performed isotype controls?
5.) In general, increasing the primary concentration might also help. I think you have already done this. In general, if there is no staining, I would rather recommend to incubate the primary antibodies overnight at 4°C as you did.
I look forward to hear back from you with your thoughts. I hope that some of the suggestions will help to improve or identify the problem.
Abcam Scientific Support
Answered on Sep 05 2012