Hi. I've recently ran few protein samples alongside with pre-stained ladder on a SDS-PAGE. Everything looks fine on the gel. Then, I did the transfer onto PVDF membrane for protein detection. All my proteins were successfully detected. But, the ladder gone missing after transferring them on PVDF membrane. Any suggestion what had happened?
I loaded 3ul of pre-stained molecular ladder on the gel. It was separated nicely and visible on the SDS gel. Before transferring, I pre-wet the PVDF membrane was with methanol. The whole transfer was conducted with 20V for 60 minutes at 4 degree.
After the transfer, you could not see the ladder on the PVDF membrane, however was the ladder still visible in the gel? You can increase the transfer voltage & run for 90mins instead of 60mins if the problem persists.
Hi, @Tushar Chakraborty . The ladder was not visible on both the membrane and gel. I wonder where it went.
The ladder might not have transferred well enough to be visible on the PVDF membrane. Apply more ladder (5-6ul) for thicker (>1.5 mm) or larger gel and then perform the same for 90mins at 20-30V.
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