Hello Everyone

I have tried to solve this problem over 2 months. My experiment is to see the inhibition of STAT3 Phosphorylation with my new drug.

Usually I can detect the p-STAT3 in the first 2 rounds of primary antibody (#9145s, 1:1000 dilution to 5% BSA TBST solution). But the signal is not strong (Signal around 4000 for MDA-MB-231 and 8000 for MCF-7) even though I stimulated the cell with 100ng/ml IL-6 for 30mins. Then the signal gets weaker or nothing when I used it for the 3rd time (I stored it with 0.05% sodium azide under 4 degree).

The following are my questions:

1. Does any one know the stability of p-stat3 antibody 9145s from cell signaling? (I did 1:1000 dilution following by 1 time freeze thaw, and the antibody gave me no signal at all. I did 4°C storage but it seems to be inactive within a week). Do you have any suggestions?

2. Is the starvation process necessary for IL-6 stimulation? If it is, usually how long it might take? (I did the overnight starvation but the signal is still weak)

3. When you add with inhibitor, how long do you usually add prior to IL-6?

4. Before you lysis the cells, do you wash with 1X cold PBS for 1 time or 3 times?

5. Do you vortex cell lysate after harvesting? (I did 15s vortex+15s sonication)

6. Boil the sample (with Laemmli buffer) to 95°C before or after centrifuge? (I did boiling after centrifuge, I usually boil it right before loading.

7. Centrifuge 14000rpm for 10mins under 4°C is fine or too harsh?

8. Do you do BCA and sample loading the same day or in 2 days?

Might sample be stored in ice for 3 hours too long (I just concern that Cell lysis+sonication+centrifuge+BCA+Boiling+loading might be too long for sample to stay in ice)

9. The MDA-MB-231 p-stat3 signal is weaker than MCF-7, is it true?

10. I can hardly see inhibition on control drug (pyrimethamine, 50uM) inhibition behavior towards p-STAT3. And that is a known STAT3 phosphorylation inhibitor, does it mean that my drug incubation in the cell not long enough? (I did 4 hour incubation)

I know I have too many questions. If you would be able to answer couple of them I will be so grateful. Thanks!