Problem of spontaneous MAPK detection from Western Bolt - (Apr/04/2006 )
I am working on XS52 cell line (dendrites cell line) for p-38, Erk1/2, and SAPK/JNK MAPK. I got an activation of p-38 and Erk1/2 at 0 minute and the bands faint away when time increase (15’ 30’ 60’). I got the activation bands even on “no stimulation” sample. However, the SAPK/JANK is fine. It activated at 15’ and 30’ on the positive control. I try serum withdraw and resting the cell in 37C incubator for 5 hours on RAW 264.7 cells, and the “no stimulation” still activate the p-38 and Erk1/2. I am wondering if any of you had this problem before and any suggestion for me to try? Please help!!!! I am stuck!!!
well, I can tell you that there's usually a baseline level; so the band for the t0 samples shouldn't concern you. there's always going to be a baseline, do you see?
however, the fact that the p38 and ERK REDUCED in the first 30 minutes with your stimulation is not so great
if you have the cells, I would repeat your timecourse at 3-5 minute-intervals to be sure it hasn't peaked by 15 min...although that does not seem to be the likely problem, it is something you might want to check? how do your protocol and expression pattern compare with published stuff for your cell line? I don't see why your p38 and ERK would be less after induction...it doesn't make sense, unless another pathway is triggered which is turning them off...which still doesn't make sense because you're doing known positive controls...
Thanks for replying so fast.
Well, I have been questioning myself of the same questions you point out and that's why it is frustrating me now. The problem is that nothing make sense in the results. I am attaching the result pictures to this reply. Please take a look. I did the p-JNK in the beginning and then I stripped them and did the p-Erk1/2 and then p-38-P. So, these 3 Abs are performed on the same blot.
I am thinking to try again on serum withdraw and resting the cell for a longer time. Hopefully, it will solve the problem. Please let me know if you have any idea.
Thank you so much.
can you tell me the way you are depriving the cells of serum?
Well, I just wash the cells twice with normal saline, then, I put them in plain RPMI for resting 5 hours before I stimulate them. Any idea?
that appears to the opposite of what I would expect (I would expect that to be the pattern for your non-phosporylated control)
do you have a sample-loading control? are you certain you're adding the same amount of protein to each well? ...although I suspect this is not the problem it needs to be ruled out
one other possibility...somehow did something happen during stripping to remove some of the protein in an uneven pattern across the membrane? also a long shot, but I'm struggling with this one, it doesn't make sense to me either..and it explains why the JNK looks right, since you did that prior to stripping
scarlett i suspect your media. your cells might be getting stimulated some way through other pathways. i donot see any problem with protein loading or stipping either.
some times this kind of problems arise if the cells you are using are continuously cultured. usually people tend to discard their cell line after a certain no. of generations and use freshly thawed cells.
try using freshly thawed cells, it might solve your problem
any way all the best
i can just suggest you that, if you pippette cells very rapidly, then it may activate stress response by which u may expect some activation at o time point.
make sure that while inducing cells, u will not activate these stress related activation of MAPks.
hope this helps
I got an activation of p-38 and Erk1/2 at 0 minute and the bands faint away when time increase (15’ 30’ 60’).
Thank you all for your advise. I just finished another blot today and now I am pretty sure the XS52 cell lines are sponteously express Erk1/2. This time, the signal didn't go away after 0' time point. I washed the cells with Normal Saline and resting them in normal saline for 5 hours before I stimulated the cells. I only take out one set samples for one time point stimulation and I centrifuged the cells in 4C. I took one sample right after I scraped the cells from culture. The signal is very strong. So, scarping the cells probably activate the MAPK. Now, the only thing I feel strange is that the LPS didn't activate the cells. I am wondering if anyone did the MAPK activation on DC by LPS (the bands intensity are all the same in NS or LPS in all the different time points)? If so, would you like to tell me how you do it? Thank you very much!!!
I took one sample right after I scraped the cells from culture. The signal is very strong. So, scarping the cells probably activate the MAPK
before you scrape out cells u add lysis buffer or some other buffer for protien stability. in this case u donot have to worry too much about scraping cells coz scraping cell will take one minute or less.
moreover when u scrape cells u should keep ur dishes on ice. so u can make sure that on ice most of the enzymes does not get activated.