For the best experience on the Abcam website please upgrade to a modern browser such as Google Chrome
If you continue without changing your cookie settings, we'll assume you’re happy with this.
LOT NUMBER GR-42219 ORDER NUMBER 911977 DESCRIPTION OF THE PROBLEM No signal only in the mouse sample, good signal in human samples SAMPLE mouse liver homogenate and cell extracts from hepatoma cell lines PRIMARY ANTIBODY ab24434, dilution 1:1000 in milk 5% in TBS+Tween 0.1%, overnight (15 hours) at 4C, 3 washes 10 min each DETECTION METHOD Immobilon Western Chemiluminescent HRP Substrate (Millipore cat no. WBKLS0100) POSITIVE AND NEGATIVE CONTROLS USED positive control: mouse liver homogenate, negative control Huh-7 cell line ANTIBODY STORAGE CONDITIONS aliquoted and stored -20C SAMPLE PREPARATION Lysis buffer (liver): NaCl 150mM, TRIS 10 mM, EDTA 1 mM, EGTA 1 mM, Tryton X100 2% (pH 7.4)+ Protease inhibitor cocktail (sigma P8340) Lysis buffer (cells): NaCl 150mM, TRIS 50 mM, sodium deoxycholate 0.1%, SDS 0.5%, NP-40 1% (pH 7.5)+ Protease inhibitor cocktail (sigma P8340) AMOUNT OF PROTEIN LOADED 30 microg ELECTROPHORESIS/GEL CONDITIONS SDS PAGE Tris-Glycine, 12% Acrylamide TRANSFER AND BLOCKING CONDITIONS transfer Tris-glycine-20% methanol for 90 minutes at 0.3 A Blocking Milk 10% in TBS+Tween 0.1% for 60 minutes at RT SECONDARY ANTIBODY anti rabbit IgG-HRP (R&D cat no. HAF008), dilution 1:5000 in 5% milk in TBS+Tween 0.1%, 90 minutes at RT, 3 washes 10 min each HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 1 HAVE YOU RUN A "NO PRIMARY" CONTROL? No
Asked on Aug 11 2011
Thank you for your enquiry regarding ab24434 and for taking the time to provide some useful details of the experiments. I am very sorry to hear that you are having problems with this antibody. The antibody is working well with HuH7 cell line, which means there is no problem with the antibody itself. I presume there might be a problem with the preparation of tissue lysates. It would help me in understanding the fault if you could send the protocol used for preparation of tissue lysates and WB image? Looking forward to hearing from you soon.
Answered on Aug 11 2011