Problem

After Transferring the membrane picture with Ponceau S Staining looks like the membrane is burning and has poor transfer. Can someone help with this?

I also noticed something weird on the gel after the transfer, it seems there are some blue spots with Coomassie Blue staining.

Gel condition

12% gel

I load 30 ug protein in each lane with six samples and two ladders separately.

Transfer condition

Wet method: 19-hour/constant 30V at working fridge (4 degrees) with ice bucket

PVDF membrane active with the methanol > 1 mins

The transfer buffer is fresh with 25 mM Tris, 192 mM glycine, and 20%methaol but make a 10* stock solution of transfer buffer, which is 250mM Tris 1920mP Glycine, then add 700ml ddh20 and 200ml Methanol to make 1L transfer buffer.

Picture

1. Picture with PVDF membrane after Ponceau S Staining

2. gel with Coomassie Blue staining after transfer

3. sandwich wet method: only show sponge/ two filter paper/ gel/ (start from black Sponge two filter paper, gel, membrane, two filter paper, Sponge)

4. gel after electrophoresis.