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Hello again, I did load 10 micro liters of Daudi cell lysate. As far as I understand it does correspond to 20 micro grams of the protein load. There is a band using BIRC3 monoclonal antibody which I think corresponds to the protein, but there were no bands on HELA and A431 cells lysates. On the other hand there were very faint bands using polyclonal but unlike the monoclonal antibody, polyclonal did show some bands in the HELA, A431 as well as Daudi cell lysates.Although Daudi cell lysates showed several bands. Two of those were too close to the real band. I dont really know what is the reason. I used 5% BSA to block the membrane and then I diluted my antibodies in the PBS-Tween 0,1%. I incubated overnight. ECL kit is new. I used it second time during this experiment. I also use fluorescence antibodies and tried those but unfortunately didnt get anything either. I still have those membranes and can try to run the GAPDH to make sure that the ECL is fine. ALthough people in our lab used that kit afterwards and didnt have any problems. Refund sounds great, but I would rather get any signal since this is a very critical experiment in my project. I need to get any data and if you can help me fix this issue that would be fantastic. Thank You
Asked on Dec 09 2011
Thank you for your reply and for sending the additional information. I am in touch with the testing laboratory to ask for further suggestions to get the correct results. I do have a few more questions, just to make sure I understand the images that were sent. Could you possibly label the molecular weights of the ladder? And also to clarify, the left membrane with the high background is the monoclonal or the polyclonal? I apologize for the inconvenience but I have conflicting information in my notes and I want to make sure the details are accurate. I will get back to you once I hear from the lab, and please let me know if you have any questions. I look forward to hearing from you and resolving this promptly.
Answered on Dec 09 2011