I am new in WB and hope to get some helps from you guys in optimizing my runs. Thanks.
I am getting a SDS-page like picture in a PVDF blot, which is very unusual. Please have a look of the attached files. Do you have any idea on what went wrong with the runs? The ladder is a prestain ladder so it shouldn't be detected by a chemiluminescene system but I got it in my gel picture which is weird to me. I have tried to increase the washing time and tween buffer concentration in the wash buffer in order to reduce the background signal but it doesn't really work.
Following is the information of the runs.
1. Membrane used = PVDF membrane (pretreat with methanol for a few seconds before soak it in transfer buffer)
2. Detection system used = GAM HRP Detection System
3. Primary Antibody Dilution: 1:2,500
4. Secondary Antibodry Dilution 1:10,000
5. Expected protein size: 92 kDa
6. Transfer method: Semi-dry transfer with 48mA for 1 hr 45 mins.