Does anyone know a good positive control (treatment) for ERK activation in MDA MB-231 cells?
Thank you in advance.
i have looked at phosph erk many times in the past via western blot in rat kidney cortex. I purchase the antibody from cell signaling. It is a really good antibody and is specific.
http://www.cellsignal.com/product/productDetail.jsp?productId=9101
The most common positive control is Jurkat + PMA Cell Lysate. I have included a link below for the santa cruz antibody and it lists at the bottom if you scroll down the positive controls for this antibody. I am not sure if you can treat the MB-231 cells with PMA, but that is the most common positive control for erk phosphorylation.
http://www.scbt.com/datasheet-7383.html
Thank you for the suggestion, do you know the specific protocol concerning PMA treatment? I suppose it is a short one, 15-60 min?
Hi Josip,
in my experience, MDA-MB-231 already have quite high levels of p-ERK in steady state (and even upon starvation for 24h) which might be due to high expression of EGFR in this cell line. What kind of experiment do you want to do?
Best,
Cindy
I am analyzing ERK activation in conditions of reduced uPA activity in MDA MB-231 cells.
Having problems getting pERK signals with Becton-Dickinson MAP Kinase Activation Sampler Kit so this is why I am looking for positive control
Okay. I think in this case I would consider a) switching to Western with a good antibody(e.g. the one suggested by Brandon) because there I'm sure you will see signals even without prior activation or b) trigger signalling with PMA or maybe EGF
Hi Josip,
In my experience, EGF stimulation of cells (~100 ng/ml) for 10 min. will give a great phospho-ERK signal. I agree with Brandon and Cindy that the Cell Signaling antibody should work really well for this, and it is the antibody I've used most frequently in the past.
Good luck!
Natalie
I am not sure of the does of PMA to use in cells. I have only used the predetermined control supernatant that can be purchased. I hope this helps.
Hi, There is another very inexpensive way to induce Phospho-ERK. Give a 3-5 min. of Potassium Chloride (KCl) 50-100mM treatment to the cells and collect. make sure you have phophatase inhibitors in your lysis buffer. I am sure you will get a very good pERK activation. I have used in various cell lines. Good Luck
Ref:
http://www.nature.com/srep/2013/130122/srep01103/full/srep01103.html?WT.ec_id=SREP-631-20130201
http://www.sciencedirect.com/science/article/pii/S0306452210002198
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