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LOT NUMBER -- NOT SPECIFIED -- ORDER NUMBER -- NOT SPECIFIED -- DESCRIPTION OF THE PROBLEM Multiple bands. The expected molecular weight is 85 kDa, whereas I got a major slower band at about 100 kDa (which I believe is the correct band) and a faster, minor band at 75 kDa. Does the protein degrades/alterates upon boiling? Samples were, as usual, boiled for 3' before loading. SAMPLE Mouse brain extract. PRIMARY ANTIBODY AbCam ab951 DETECTION METHOD ECL and X-ray film ANTIBODY STORAGE CONDITIONS Antibody was supplied already dissolved and was stored at 4°C, as indicated. SAMPLE PREPARATION Fractionated extraction using: Fraction 1) Tris-HCl buffer, aprotinin, leupeptin, NP40, SDS, PMSF, Na3VO4, EDTA. Fraction 2) TNT buffer Fraction 3) Same as buffer 1, but with the addition of deoxycolate and EGTA in place of EDTA. AMOUNT OF PROTEIN LOADED 10 micrograms. ELECTROPHORESIS/GEL CONDITIONS Reducing 4-12% Bis-Tris gel, XT-MES BioRad buffer. TRANSFER AND BLOCKING CONDITIONS Used the TurboBlot transfer system by BioRad on nitrocellulose membrane. Blocking 4% non-fat dry milk in 0.2% Tween-TBS for 2h at R/T. SECONDARY ANTIBODY Jackson HRP-conjugated goat anti-mouse 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
Asked on Feb 23 2012
Thank you for contacting Abcam.
I think you are correct that the band that you are seeing at 100kDa is correct as based on the Swissprot sequence http://www.uniprot.org/uniprot/Q61391, there are q few glycosylation sites on this protein that would account for the increase in the predicted molecular weight. The 75kDa mat be a non-specific band in which case changing your blocking reagent to 5%BSA may help to reduce that signal. Otherwise if it is a breakdown product then you could increase the amount of protease inhibitors that you are using.
The antibody is covered under our Abpromise for six months and is guaranteed to work in western blot on mouse samples . If we cannot resolve the issue you are having with the antibody then I would be happy to either send a replacement antibody or to process a refund.
Please let me know if there is anything else I can help you with.
回复于 Feb 23 2012