We are trying to determine efficiency of a specific GEF in activating Rac in prostate cancer cell lines. Serum alone, or EGF alone are not doing the trick. Has anyone done this already?
Hi, What concentration's of EGF you have tried and the time points ?? What assay are you going to use for Rac activity?? If you can answer these, may be i can help you :)
We use a Rac activity pull down assay with GST-PBD. I am using EGF at 100ng/ml at 15, 30 and 2 hour time points. I am trying a combination of EGF(100ng/ml) and IGF(10ng/ml) now and will see if that helps.
I suggest you to check at 1, 5 and 10 mins. Dose is fine. Most probably you will see 2-3 fold at 1 and 5 mins. Best of luck
Would be better to serum starve the cells for 12 hrs before stimulation and shorter time points (1-10 min) would give you better result.
PDGF at 20 ng/ml should work fine. However the activation occurs very fast (within 30 sec to 1 min). Same for EGF, so you should make sure you perform your assays extremely fast. After approx 5 min, the activation is gone.
Yes, you should definitely starve (how long will depend on the cell line you are using)
Among growth factors, PDGF is the best to activate Rac. You need to make sure if the cancer cells you used have high expression of PDGFR. Another method is to activate Rac by cell adhesion. Resuspend the cells and replate them on coated plates. Rac would be activated during the cell spreading process.
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