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I am trying to get a WB of endogenous DGAT1. This is my first time using your antibody. Starting material was WT and DGAT1-KO mouse tissue loading 30 micrograms of protein. 4-12% gradient gel with MOPS running buffer 200V for 1hr. Transfer onto nitrocellulose in Tris-Glycine buffer 20% methanol at 75V for 2.5 hrs. I blocked with 5% fat free milk in TBS with 0.1% tween-20. Antibody was incubated for 1hr at RT in the blocking buffer at 1 microgram/mL. The secondary antibody was diluted in blocking buffer at 1:10,000 from 1mg/mL stock and incubated 1hr at RT. Detection with ECL reagents. In between primary and secondary, I did 3x 10 minute washes with TBST, and 3x 10 minute washes after secondary before detection. The bands I see are totally aspecific and there are no differences between WT (lane 1) and DGAT1-KO (Lane 2) bands. I included a cell line over-expressing human DGAT1 as a positive control (Lane 3) and also saw no specific detection there. Please advise.
Asked on Jul 12 2012
Thank you for contacting Abcam regarding ab59034.
I am sorry that this antibody is not working for you in WB. I have reviewed the protocol information and data you sent and I agree that this product is not performing as stated on the datasheet. I am happy to offer a replacement or credit, per our Abpromise guarantee. Please let me knowwhich you would prefer.
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 Jul 12 2012