Hello,
I been running experiments to test the effect of an antibody on Multiple Myeloma cells, which involves the SDF-1 effects on AKT, in every paper I've read they successfully have phosphorylation of AKT by using SDF-1, but I can't detect any band besides my loading control, I haven't tried total AKT.
The methodology of my experiment is as follow:
Cells were cultured with RPMI media 10% FBS at 37 °C 5% CO2 until reached desired confluence. For each sample 3x106 cells were seeded into 24 well plates and starved overnight with 1% FBS media at 37 °C 5% CO2. Cells were cultivated with 30 nM SDf-1 for 1, 5 and 10 minutes, along with one control sample with no SDf-1; as well cells where cultivated with specific antibody for 15 minutes and then with 30 nM SDf-1 for 1, 5 and 10 minutes. Cells where centrifuged and lysed with Pierce™ RIPA buffer (Thermo Scientific, 89901) complemented with Protease Inhibitor Cocktail cOmplete™ (Sigma, 5892791001) and the phosphatase inhibitors cocktail following product instructions.
I loaded 40 ug of total protein in to the SDS gels, and then transfer by western blot (iBlot). The membrane was blocked with PBST 5% milk for 1 hr, and then washed with PBST 3 times 5 minutes each time, then incubated over nigth with anti-p-AkT (I have tried ser427 and Thr308). Membrane was washed with PBST 3 times 5 minutes each time and then incubated with secondary HRP antibody for one hour and washed with PBST 3 times 5 minutes each time.
So if anyone has any suggestions I'll be thankful,
When we do western blots for phosphoproteins, we have had much better luck using 2.5% BSA as a blocker rather than milk, finding that milk interferes with binding of some anti-phosphoprotein antibodies. Our best protocols also include 0.1 mM Sodium Orthovanadate (to inhibit potential phosphatases) and 0.05% Tween 20.
If this doesn't work, then try a cell type that is known to have a robust AKT response and stimulate with insulin. That would be a good positive control.
Polina Weitzenfeld thanks a lot.
Yes I've detected CXCR4 in these cells by FACS, and I have done some functional Chemotaxis migration assays with SDF-1 (dose response curve and everything) and they responded to SDF-1, but in the westerns I haven't have any luck, but I'll try steps you mention as adding cold PBS after the five minutes.
very useful information
To look at phosphorylation of proteins in adherent cells, we simply rinse once with ice-cold PBS and then add boiling hot RIPA buffer (look it up) and scrape everything off the plate into a microfuge tube and boil it. This stops all enzymatic activity and dissolves the cells. The solution will be viscous because of the released DNA, so you need to pipette the solution repeatedly to shear the DNA before loading onto the separation gel.
Henry Steven Wiley thanks, very useful info, for now I'm using non adherent cells, but I might try with some adherent cells lines that I have.
Polina Weitzenfeld Thanks a lot.
the cells I'm working on are on suspension.
Other question, do you starved the cells? I've been starving the cells (overnight free serum media) before the experiment.
@polina weitzenfeld when you add the SDf-1 do you added in media (free serum) or PBS?
I'm having this results, don't know why, these is the P-AKT, when I look for the loading control or the total AKT, it looks fine ( I'm using TBST 0.1% and BSA 5%)
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