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Thank you for your quick reply. The antibodies which did not function were: ab3484-100 (lot:52477) and ab8934-100 (lot: 44507). I used the antibodies in western blotting, diluted at 1:500 in 5%milk-0.05%Tween-PBS. Secondary antibodies were anti-rabbit(IgG)-HRP (Chemicon, has worked constantly in our lab)at 1:5000 dilution in 5%milk-0.05%Tween-PBS. The samples were mouse primary hepatocyte cell lysates (lysis buffer: 150 mM NaCl, 50 mM HEPES, 5mM EDTA, 0.1% NP-40 + protease/phosphatase inhibitors)which to my knowledge should contain large amounts of PPARalpha. Due to this fact, here were no positive controls (and we had no clue what would be a better positive control then hepatocyte lysates). I hope this is of some help to clear up the issue as these antibodies are critical in our studies.
Asked on Oct 25 2004
Thank you for your email and the details in which you have provided. Can you please tell me - what exactly are you seeing on your blots? Are you not getting any signal at all or are you seeing bands but not at the correct size? How much protein did you load? Did you try increasing the concentration of the primary antibodies? Thank you, and I look forward to hearing from you.
Answered on Oct 28 2004