MAP Kinase study - (Mar/23/2006 )

hi friends ! iam working on MAPK (p38, Erk 1/2) activation patterns in macrophages after stimulation with different ligand. iam using lysis buffer ( tris 25 mM pH 7.4, NaCl 150mM, NaOVa 1mM, NaF 10mM, EDTA 1mM, EGTA 2mM, Leupeptin 50ug, Aprotinin 50ug, Pepstatin A 50ug, PMSF 1mM, DTT 2mM, IGEPAL 1% v/v), but could not able to spot out the phosphorylated forms of the MAP Kinases. Antibodies iam using are polyclonal and one of my buddies is getting results using the same antibodies. he suggested me few points but they all failed. iam using Amershams advanced chemileuminicence kit for detection. kit is working fine with other antibodies. protein amount loaded is 50ug/well. also iam getting lot of non specific bands (but not the phosphorylated forms)
can any one suggest
1. lysis buffer , protease inhibitors
2. stimulation time with the ligands to spot phosphorylated forms.
3. amount of protein to be loaded to get clear picture ( as i feel that iam loading too small quantity to spot phosphorylated forms)
looking farward for some positive responses

Hey Shiva,

You might want to check the confluency of your cells. I dont think you mentioned the type of cells u r using...but i have seen that with soem cell types...how confluent your flask is determines the intensity of the signal. I know ppl say that you have to use a subconfluent flask to get good kinase activation. However, my experience is that the cells need to be a little more than subconfluent but not over confluent. I have worked on this independently and one of the guys at cell signalling agree with me as he was helping me trouble shoot this horrendous problem that I was having.

Another thing i was told on this forum and it worked was to use fresh transfer buffer. So i normally make mine when I am running the gel, stick it in -20 20 mins for it to cool down and use it for my transfer. knock on wood but it works....

I dont think you should really worry about the chemi and yes with human cell lines I use anywhere between 20-50 ug of protein and it works.

One other aspect that you might want to consider is sonication vs lysis. I have seen that if you sonicate cells ( i use the same lysis buffer as you) then the ERK comes as one band or the 42 band is much lower than expected. However, if i lyse my cells by vortexing the cells then my erk bands come out nice and close to each other.

good luck and let me know how things work out...

I agree completely with Casper.
I wanted to ask more details about blotting.
what kind of Ab are you using? anti-phospho tyrosine? or anti-phospho MAPKs? (anti active ERK from promega works very well :blocking in TBS plus 3 % BSA, wash in TBS plus 0.05% tween-20 (TBS-T). incubate antibodies in TBS-T plus 0.1 % BSA.)

thank you casper and missele for your promt responses. i think or may be i got the answer. i am using 92-95% confluncy. my work is related with pathogen (candida) associated immunomodulation. candida sticks itself to the plate and to prevent it iam using high confluency.
i have also observed that high confluncy hinders phagocytosis obvious by the decrease in the no. of phagocytosed cells.
iam using anti phospho MAP kinases and the cell line used is mouse Balb/c derived J774 macrophage cell line.
i block the PVDF membrane with 5% BSA . i use 0.1% T-20 with TBS as recommended by the Amersham kit and it works fine for other antibodies
i donot think there is any problem with the transfer as i usually prepare fresh transfer buffer and transfer is done in a refrigirator at constant voltage (20 V for 14 Hrs)
thank you very much for your suggestions. iam a gainer.