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DESCRIPTION OF THE PROBLEM No signal SAMPLE Neurodifferentiated human embryonic stem cells PRIMARY ANTIBODY Abcam (ab26113), Mouse monoclonal to GAD65, in Knouckout Serum replacement @ 1:500, incubate for 1 hour and wash 3 times with 100 uL PBS ++ DETECTION METHOD GE Healthcare In Cell 6000, High content analyzer using confocal imaging POSITIVE AND NEGATIVE CONTROLS USED As this is a routine procedure, we do not use positive and negative controls on every plate. This assay plate was stained for other markers which worked fine indicating that there was no problem with the staining method or the image capture system. We have previously used this antibody in this manner and have gotten good results. We expect our cultures to be 60-70% GAD65 positive. We have in the past run a negative control (undifferentiated stem cells). Previous run (GAD65) in red ANTIBODY STORAGE CONDITIONS stored at -20C in 10 uL aliquots FIXATION OF SAMPLE 10% formalin (Sigma-Aldrich ANTIGEN RETRIEVAL None PERMEABILIZATION STEP Yes, 0.25% triton x-100 BLOCKING CONDITIONS No blocking SECONDARY ANTIBODY Invitrogen (A31571), Donkey anti-mouse IgG Alexa Fluor 647 , in Knouckout Serum replacement @ 1:500, incubate for 1 hour and wash 3 times with 100 uL PBS ++ HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 3 HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes
Asked on Feb 14 2012
Thank you for contacting Abcam.
Based on the protocol the customer sent, there are a few suggestions that may help resolve the issue:
1 - Increase the concentration of primary antibody used to 1/200.
2 - Increase the incubation time to 3 hours at room temperature.
I understand that the customer had previously purchased vials of this antibody and it has proved to be successful for them but as we have not tested ab26113 in either human cells or if it is not covered under our Abpromise. Please let me know if changing the above does not prove to be successful and I will if there is anything else I can do.
Answered on Feb 15 2012