Anti-PPAR alpha (phospho S12) antibody (ab3484)

Follow up to previous correspondance:

Thank you for your suggestions. I was able to get a signal with all of the antibodies, but I am worried about the sizes of the bands. According to your data sheet, the size of ALL the bands is about 52 kD. However, only the PPARalpha (ab8934) detects a specific band of this size. The bands of the phosphorylated forms of PPARalpha (antibodies ab3484 and ab3485)are closer to 70 kD, but are quite specific, producing little background except during long exposures (1h). Are you completely sure that the sizes of the phosphorylated forms should be approx. 52 kD, or is it possible that they are larger, i.e. the 70 kD bands? I hope you are able to answer this last query, as I want to ascertain that the signal we are getting is the proper one. For you question concerning storage, the antibodies were aliquoted and stored in -20 C immediately upon arrival.

ANSWER:

Thank you for your recent e-mail. My colleague Jennifer is unexpectedly away this week and I would like to help you. I understand you have recurring problems with ab3484 and ab3485 detecting the wrong bands and your protocol seems fine.

I would like to send you replacements for these two antibodies as they should recognise well the 52kDa band.

Can you please provide me with the order number used to purchase the antibodies and I will arrange replacements to be sent to you ASAP.

I'm very sorry for the delay, thank you for your patience.

I look forward to hearing from you soon,

Thank you for your mail. With the anti-PPARalpha (ab3484) we did not see any signal. With anti-PPARalpha (ab8934) we saw various background bands after 1 h exposure, but not one of the correct size. We loaded 60 micrograms of protein/lane. We did not try increasing the concentration of the primary antibodies, as 1:500 is already very high. Hope this is of some help.

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.

ANSWER:

Thanks again for your email. At this point I would like to make the following suggestions that will hopefully help you out. For ab8934 I suggest using 3T3 whole cell lysate as a positive control. For ab3484, I suggest increasing the concentration of the primary and incubating overnight at 4C. If you still do not see a signal, I will send you a replacement vial free of charge.

For both, how were the antibodies stored after you received them?

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.

ANSWER:

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.