Question (14934) | Rabbit Anti-Mouse IgG H&L (Alkaline Phosphatase) (ab6729)

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Question

BATCH NUMBER 131853 ORDER NUMBER MBPO/023 DESCRIPTION OF THE PROBLEM No signal of the relavant antigen (I have tried two different primary: see below), many non-target bands. SAMPLE Budding yeast cell extract (PCNA-Myc and non-tagged). PRIMARY ANTIBODY Abcam ab56 anti-Myc (9E11), Lot 130523, 1000x to 5000x dilution, 1 hr. or Abcam ab29 anti-PCNA, Lot 119707, 1000x to 5000x dilution, 1 hr. DETECTION METHOD CDP-star reagent from Perkin Elmer. POSITIVE AND NEGATIVE CONTROLS USED For detection of Myc-tagged protein, I included non-taggad sample as negative control. Size difference in Myc-tagged and non-tagged samples must help to identify correct band. Same blot has worked fine with HRP-Anti Myc from Santa Cruz, enabled me to compare the result. ANTIBODY STORAGE CONDITIONS In fridge (4 degree) w/o dilution. SAMPLE PREPARATION Roche Complete as protease inhibitor. Boiled soon after addition of SDS. AMOUNT OF PROTEIN LOADED up to 10 ug. ELECTROPHORESIS/GEL CONDITIONS Reducing, mainly 6% and 12%. TRANSFER AND BLOCKING CONDITIONS 5% milk in PBS-T as blocking. SECONDARY ANTIBODY ab6729 HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 6 HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes WHAT STEPS HAVE YOU ALTERED? I tried several different dilution of both primary and secondary (x1000 to x5000). At 1000x dilution, backgorund was very high. At 3000x or 5000x dilution, background was reduced, but no target band. Similar pattern of background bands. I also tried "No Primay". It gave quite similar background bands. ADDITIONAL NOTES I used ab6729 in combination with ab29 anti-PCNA and ab56 anti-Myc. Both gave essentially identical result (no target band, many background). The same blot worked fine for HRP-anti Myc from Santa Cruz. Also not tested in western, I believe that ab56 anti-Myc is OK, because I and a student have used it for ChIP and got nice results. Taken together, I conclude that an6729 doesn't specifically recognize mouse IgG. I prefer replacement to other antibody with good record, such as ab6790, rather than refunding.

Answer

Thank you for contacting us for technical support with ab6729. I think the problem you are experiencing is due to the fact that budding yeast expresses an endogenous form of alkaline phosphate (http://www.expasy.ch/uniprot/O60109) and by using a secondary antibody conjugated to AP this creates a background signal. I would therefore recommend using a HRP conjugated secondary antibody (and ECl + detection kit) to prevent this problem. Please do not hesitate to contact us again if you still experience problems with an HRP-conjugated secondary antibody,

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