No bands seen when probing for P-JNK - (Oct/10/2007 )

Hello everyone,

I am trying to probe for P-JNK using homogenized (frozen) liver tissue. I nor anybody in my lab has experience working with P-JNK or lysates from tissue. Therefore, I have looked for suggestions in the literature but am pretty clueless as to were to start making adjustments.

Here are some details of my set up:

1- high protein concentration, loaded 100ug per well
2-Used pre-made ripa buffer (PMSF, sodium Orthovanadate and protease inhibitor from Santa Cruz) for lysis. Somebody suggested to use NP-40 when probing for phosphorylated pro. instead but I have not tried it
3- Used Hela extract TNF-a (human) as positive control
4. Used P-JNK (G-7): sc-6254 and total JNK (F-3): sc-1648 from 2006


I did not see any bands when probing for P-JNK (not even for the positive control) the first time but did see bands for total JNK. The second time I did not get any bands, even though the marker appeared, which ruled out the possibility of a transfering problem. But, then I used Ponceau and did not see any bands stained. I wonder if I am not using the proper antibody since I have been reading about anti P-JNK and Phospho -c- JNK but do not understand the difference.


Thanks in advance to anyone who can help me with this matter

have your tissues been prepared with protease inhibitors? that's the first thing -

also, follow this link to the short answer - phospho-c-Jnk is, I think, descriptive of phosporylated cJNK. P-JNK would be phosphorylated JNK. JNK is phosporylated by MEKK (at least in B-cell signaling), it in turn phosphorylates JUN

cJNK is more specific than JNK, but in the case of your antibody's name it could merely be semantics. that's the short answer - I think cJNK phosphorylates cJUN, etc. does that make sense?


to really understand the signalling pathways involved and understand what JNK is and what it does, I would recommend going to the literature. there are a number of good reviews - also, there is a ton of information on a part of pubmed called OMIM (look for it in the upper toolbar) but it has to be sifted through.

Could anyone else who has worked with JNK and tissue lysates send me some suggestions on what I should try? PLEASE!!


Thanks so much!!