Hi, I used a semi-dry system with 15V 1hr transfer time. The buffer composition was provided by the company who we bought our primary antibody from which is 1.52g Tris, 14.4g Glycine, 100ml methanol made up with distilled water to 1L. However, after I do the transfer, I could see very obvious bands on the filter paper underneath the membrane. Is this a sign of over-transfer? A staining of my gel before and after the transfer shows most of the protein has been transferred. Also, the wash buffer composition they provided is: 1X TBS, 0.1% Tween 20, 1% skimmed milk, whereas I've never used milk in my wash buffer before. Any comments?
What bands are on the filter paper? Smaller proteins are more likely to transfer through the membrane. To enhance capture of small proteins, 20 kD or less, using a membrane with .2 micron pores is better than .45, and higher methanol is helpful. If high molecular weight bands are on the filter paper, there is an obvious problem as those bind better due to more binding groups per molecule. Learned all this on a webinar yesterday.
Do the bands for your ladder/marker also appear on the filter paper underneath?. I encountered a similar problem, but in my case I added too much methanol when preparing my transfer buffer. You can also adjust the voltage and time for your transfer to check if it helps solves the problem.
- Badges
- Science topic
- Similar topics
- Gels