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DESCRIPTION OF THE PROBLEM Non-specific band Wrong band size at the site of 35kD. SAMPLE Cell extract from HepG2.2.15. PRIMARY ANTIBODY A dilution of 1:800 was used for probing with an incubation of overnight at 4℃, then washed in TBST buffer with agitation for 4 times, 10 minutes per wash. DETECTION METHOD Pierce ECL Western Blotting Substrate Visualized by Image reader (Las 3000, Fuji film). POSITIVE AND NEGATIVE CONTROLS USED No ANTIBODY STORAGE CONDITIONS -20℃. SAMPLE PREPARATION 1% NP-40 cell lysis buffer (1% NP-40, 1 mM EDTA, 50 mM NaCl, 10 mM Tris-HCl, pH 8.0). AMOUNT OF PROTEIN LOADED 30μg per well. ELECTROPHORESIS/GEL CONDITIONS Wet transfer using nitrocellulose membrane (watman) in a standard buffer with 1X Tris-glycine omiting SDS but additioning methanol to a final concentration of 20%, as discribed by Abcam Ltd. TRANSFER AND BLOCKING CONDITIONS For membrane blocking, a 5% non-fat milk solution diluted in TBST was used. SECONDARY ANTIBODY HRP-conjugated secondary antibody against mouse IgG (, diluted in TBST with a dilution of 1:5000) was used, incubated 1h at RT and washed as same as the procedure used for primary antibody. HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 2 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes
Asked on Dec 21 2011
Thank you for contacting Abcam regarding ab8637. I am sorry that you have been experiencing difficulties with this antibody in WB. I have reviewed the protocol information and image you provided and would like to ask some additional questions. There seems to be a decrease in the expression of your protein of interest in the lanes from left to right. Were these treated differently in any way? GAPDH appears consistent, but I am not sure if you have treated the cells. What percentage gel did you run? Was everything run under reduced, denatured conditions? How long did you boil the samples before loading? It is recommended to boil the samples for at least 10 minutes for this antibody to work well. Also depending on the percentage gel and sample preparation, the molecular weight may run slightly different than the predicted molecular weight. I hope these guidelines are helpful. I look forward to your reply so that I may assist you further. Please do not hesitate to contact us if you have any additional questions.
Answered on Dec 21 2011