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Question (24476) | Anti-COX IV antibody [mAbcam33985] - Mitochondrial Marker (ab33985)

LOT NUMBER GR9270-1 ORDER NUMBER PO-11512 DESCRIPTION OF THE PROBLEM The antibody should stain mitochondria, but it actually stained no-specifically to all cell membrane. SAMPLE HeLa cells, BV2 cells, C8B4 cell and C6 cells PRIMARY ANTIBODY 1ug/ml as suggested by the data sheet (ie 1000x dilution). DETECTION METHOD Fluorescence microscope equipped with high sensitivity camera. POSITIVE AND NEGATIVE CONTROLS USED Positive control was other mitochondria antibody (ab5298 for HeLa cells only). Primary and 2nd antibodies only have been performed and none showed positive staining. ANTIBODY STORAGE CONDITIONS Aliqoted and stored at -20C FIXATION OF SAMPLE Paraformaldehyde (4%) for 15 min ANTIGEN RETRIEVAL n/a PERMEABILIZATION STEP Triton X-100 at 0.2% for 20 min (it work for other antibody) BLOCKING CONDITIONS 2% BSA for >30min SECONDARY ANTIBODY Anti-mouse IgG conjugated with Dylight 594 (ordered from Abcam #an97017). HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? >4 HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes WHAT STEPS HAVE YOU ALTERED? n/a ADDITIONAL NOTES the following message from the customer: "Please change to other abcam antibody that react with human, rat and mouse. The antibody should be raised from mouse. The antibody can be used for IF and WB. Would you please inform AbCam that I want to have ab14705 MS404 (Anti-MTCO1 antibody-Mitochondria Marker (mouse monoclonal) to replace my previous antibody (ab33985)?"

Thank you for taking the time to complete our questionnaire and contact us. I am sorry to hear you have had difficulty obtaining satisfactory results from this antibody. The details you have kindly sent will provide us with vital information for our monitoring of product quality I appreciate the time you have spent in the laboratory and understand your concerns. It is regrettable the results have not been successful. As requested, we would be pleased to arrange this free of charge replacement. In order to do this, I am forwarding these details on to our distributors team who will be happy to organise this for you. Original order number: 940526 1 X vial ab33985 Anti-COX IV antibody [mAbcam33985 Please note that this free of charge replacement vial is also covered by our Abpromise guarantee. Should you still be experiencing difficulties, or if you have any further questions, please do not hesitate to let us know. I can also recommend to consider the following suggestions for any future experiments, which I hope may help improve the results. Although other antibodies have worked well using this procedure, individual antibodies can sometimes require individual optimization. 1. I can recommend to try a lower dilution of antibody to reduce any non specific staining. Try 1:2000. 2. I can suggest to increase the concentration of blocking agent to 5% BSA 3. I can recommend to try including wash steps if you have not already done so. Try PBS containing 0.2% Tween (a more gentle detergent) 4 times for 5 minutes at each appropriate wash step. Thank you for your cooperation. Should you have any further questions, please do let me know. I would like to wish the customer best of luck with their future research.