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Question (37358) | Anti-mTOR antibody (ab2732)

LOT NUMBER GR22858-3 ORDER NUMBER 987281 DESCRIPTION OF THE PROBLEM Non-specific staining in nucleus SAMPLE human cells in culture PRIMARY ANTIBODY Concentration or dilution 1:200 Diluent buffer 1% BSA in PBS Incubation time 2hr Was Buffer PBS Number of washes 3 times DETECTION METHOD Laser scanning confocal microscope (488 argon laser) POSITIVE AND NEGATIVE CONTROLS USED - ANTIBODY STORAGE CONDITIONS 4℃ FIXATION OF SAMPLE If yes: Fixative agent and concentration 4% paraformaldehyde Fixation time 10min Fixation temperature RT (20℃) ANTIGEN RETRIEVAL - PERMEABILIZATION STEP Permeabilizing agent and concentration: incubation in 0.1% triton X-100 PBS for 20 min BLOCKING CONDITIONS Concentration 1% BSA in PBS Blocking time 30min Blocking temperature RT (20℃) Endogenous peroxidases blocked? no SECONDARY ANTIBODY Species: invitrogen A11008 Isotype: IgG Reacts against: rabbit Concentration or dilution 1:1000 Diluent buffer PBS Incubation time 1hr Fluorochrome or enzyme conjugate alexa 488 Was Buffer PBS Number of washes 3 times HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 3 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes WHAT STEPS HAVE YOU ALTERED? - ADDITIONAL NOTES -

Thank you and your customer for your inquiry.

I believe that the images that your customers provided do show very nice specific staining.

According to the Uniprot homepage (http://www.uniprot.org/uniprot/P42345) mTOR is expressed in the endoplasmic reticulum membrane, the Golgi apparatus membrane, the mitochondrion outer membrane, theLysosome and the Cytoplasm. It shuttles between cytoplasm and nucleus.

If it is in the nucleus, it is located in thePML bodies which is nicely shown by your customers image.

I am happy to confirm that this antibody does not seem to exhibit any un-specific staining and that your customers results are very nice.

I hope I could reassure your customer that their results are specific. Please let me know if your customer has any other questions.