I am having a recurring issue with the incomplete transfer of protein bands from my PAGE gel to the PVDF membrane. SOME proteins transfer, but many do not. These bands that aren't transferring are prominent bands, so there has to be a significant amount of protein there. I have attached a picture (the stain-free PAGE gel is in the left and the blot is on the right. The red arrows indicate just a couple of significant bands that are missing.).
The transfer protocol is 16 hours at 35V constant, 90 amps. Transfer buffer is Towbin without SDS.
Any suggestions would be appreciated!
I assume the image on the left is total protein from the gel with UV, and the image on the right is your probed membrane? What antibody is this probed with? Regardless, a 10% loss in protein is expected when imaging a stain-free gel due to cross linking of proteins.
One thing can be too long transfer - some of the protein may have just escaped the membrane.
Or - too short transfer - but this can be easily tested - you can just stain the gel after the transfer and check if you can still see protein bands.
Short (about 20 seconds) activation of PVDF membrane in 100% methanol before the transfer also can help.
I assume your gel is not native PAGE but an SDS-PAGE? have you seen if the same happens if you electrotransfer to NC membrane instead of PVDF? hope it helps.
Hello Priscilla Barger
Thicker gels can result in incomplete transfer because thicker the gel, the farther the protein has to migrate to reach the membrane. Effective transfer of proteins out of gels up to 1.5mm thick can be achieved. Thicker gels, although having increased protein capacity, offer little advantage owing to reduced removal of proteins from the thicker gel matrix.
Also ensure sufficient contact between gel and membrane during transfer.
Make sure transfer sandwich is assembled correctly.
Avoid overheating during electro-transfer.
As Tomasz Fraczyk mentioned stain the gel with Coomassie blue after transfer is complete to determine transfer efficiency.
Best,
Modify your transfer buffer with MeOH and/or SDS...NC membrane can indicate this can be caused by the membrane material. This may be an issue depending on the antibodies you used. have you ever checked the transfer with ponceau staining.
I had better luck with Dunn's transfer buffer (DOI:10.1016/0003-2697(86)90207-1, 10 mM NaHCO3, 3 mM Na2CO3) than with the more common Towbin. Also, in my hands, tank blotters work better than semi-dry (probably because the heat distributes better). Try to stain the blot with Ponceau S (DOI:10.1016/0003-2697(86)90263-0) to see if protein transfer is complete (the stain washes off during blocking).