My question is regarding a western blot experiment that I am performing to observe pERK signaling by stimulation with VEGF ligand (VEGF165 and VEGF121) on MDAMB231 cells and SW620 cells (both known to be positive for VEGFR-1 and VEGFR-2 expression). I am using GAPDH as the loading control. In theory, I should be able to observe signaling as I am to use this as a positive control in further experiments.
- In my initial experiments, I was getting bands, although I was observing bands at similar intensity also in the untreated cells. I am testing the VEGF at increasing concentrations.
- I decided to serum-starve my cells at 1% for 4 hours and observed VEGF121 signaling was higher with increasing concentration although VEGF165 showed same signal as the untreated cells.
- Currently I have been repeating the experiment, this time, with a 16-18 hour starvation.
- First time, I got a blank blot, with no bands.
- The next time, I got very faint bands. Also, I am unable to see GAPDH expression too this time.
- I have tried the troubleshooting protocols, in terms of blocking buffer, primary antibody dilution, transfer time and protein loading.
- Does increasing starvation time affect expression of GAPDH too?
- What are some possible things I am missing here.
Please let me know if anyone has any suggestions.
it looks strange since your cells is known to express VEGFR and also i think it is a good idea to try overnight starvation and try other primary antibody
Which phospho ERK antibody are you using? I use the antibody from cell signaling cat#4370 and it works really well.
Are you making sure to include phosphotase inhibitors in your lysis buffer?
Have you tried other loading controls such as beta actin or beta tubulin? If you do not see any of those as well as not seeing GAPDH there is a transfer problem. Have you ponceaued your blots to make sure that you are transferring your protein?
Thank you for your reply Brandon A Kemp . Yes it's the same I am using too, the #4370. I am including PMSF in RIPA buffer.
Maybe I can try other loading controls. I stuck to GAPDH as its been used successfully in the lab for all blots.
Well regarding the transfer issue, I do see my ladder on the membrane and also I do get some, if not proper, very faint bands. Should that be indicative enough for transfer?
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