Hi,
I am trying to look for basal ERK phosphorylation in N2a cells. Although, I am able to detect very good intensity of bands for total ERK, but the p-ERK bands are hardly visible. Also, when the basal p-ERK is very low in these cells, the extent of effect of stimulation with any agonist or chemical is very minimal.
I have to repeatedly thaw new vials every week to see which batch of cells give decent p-ERK basal levels.
Is this problem faced by other members here? How can one resolve this issue such that the cells maintain good levels of basal ERK phoshphorylation?
Media used: DMEM+ 10% FBS+ Penicillin/ Streptomycin
Antibodies: p-ERK and t-ERK antibodies from Cell Signaling
Looking forward to your helpful suggestions
Apurva
How are you extracting the protein? For important tips see e.g.
https://www.researchgate.net/post/Which_cell_lysis_buffer_recipe_is_best_for_phosphorylated_proteins
Hello Agrawal,
I agree Sabine Strehl about isolation of phospho protein issue. And I'd suggest you to
- increase loading concentraion of your protein
- add more antibody (1:500 instead of 1:1000 etc.)
- use more chemiluminescence buffer or adjust waiting time (more or less) in clb.
Also, just for an idea, maybe phosphorylation affect of protein during transfer. So you can try optimize the transfer time. And lastly, if you have materials you can do immunoprecipitation. I hope you can solve problem.
Good luck!
...just to add to the above answers: 1) try to avoid milk powder for blocking membranes for pERK WB (use albumin fraction V or just Tween). 2) If you are using the same membrane for detection of both total ERK and pERK, try to detect pERK FIRST (before stripping the membrane for subsequent analysis of total ERK). Membrane stripping may affect pERK detectability. Also, you may try to find a "strong" agent that induces phosphorylation of ERK in your cells (for a positive control).
Best of luck.
Thank you all for your kind suggestions.
@Gediminas: I am going to try some of the above mentioned suggestions. But is there any way by which inherent basal level of ERK phosphorylation can be elevated so that they can be very well detected. The problem I am facing here is the levels are not at all detectable.
We use 5% BSA. Also, our lab has been for long working on p-ERK and t-ERK from rat brain samples. The pERK antibody works like a charm when the blots are probed with it after stripping the t-ERK antibody.
I am looking for a way by which basal levels can be elevated. Also, when the basal p-ERK is very low in these cells, the extent of effect of stimulation with any agonist or chemical is very minimal.
It's not that I never observed good pERK levels in these cells. It is varying with every batch of cells that I thaw. Although the media, FBS concentration, subculture method, everything is same.
.....interesting! Have you tried to assess in parallel phosphorylation levels of other MAPKs (e.g. p38, JNK)? I am just wandering if depending on the cell batch the overall MAPK response(s) may be suppressed, which would suggest that something "global" is happening to your cells during freezing/thawing cycles. Also (please do not take it personally), but make sure you have good and working phosphatase inhibitors in all buffers starting with the cell washing step just before cell lysis in SDS sample buffer.
- Badges
- Science method