Sudden Trouble with PVDF Transfer - (Jun/11/2013 )
As most of you are I am desparate for advice!
I have been performing western blots using PVDF, however, recently I have noticed bad transfer. I can tell this because both my ladder is not noticable and Ponceau S Staining of the membrane looks terrible.
I have stained my gels with Coomassie, and my proteins are clearly transfered and OUT of my gel (8% SDS-PAG), but they are not on my membrane? After a series of experiments, when I tried a nitrocellulose membrane things looked great. Both my ladder and protein stained with Ponceau looked good. We recently ordered PVDF from another source, however again this did not look good.
We will switch to nitrocellulose if we have to, but it is SO odd that PVDF used to work for me, but is not. Any ideas???
I do pre-wet my PVDF membranes accordingly (100% MeOH, then transfer buffer). I have double membraned my blots and nothing is going through. It's almost like my meembranes just aren't "sticking" to my PVDF anymore... what is going on?
I am in my final year of my doctorate, so I have been doing westerns on and off for 6 years... I have seriously tried everything I can think of... Please help!
Thanks so much!
Sounds like something to do with either the PVDF or the methanol - are you using electrophoresis or higher grade methanol? Bulk methanol often contains impurities that could (potentially at least) interfere with the charge on the membrane.
Thanks so much. I thought something might be wrong with my PVDF so we just ordered new membrane from another reliable source.
I will look into our MeOH and figure out if I need to get a higher grade!
Thanks so much! It is new MeOH, but like you suggested maybe its impure!
Thanks! And please keep the ideas coming
- I actually just checked and are MeOH seems okay is Reagent grade ACS
maybe too little methanol in the transfer buffer.
what are the pore sizes of the pvdf and the nitrocellulose?
I've used both 0.45 and 0.2 um PVDF and I can't find it on our nitrocellulose (that's working) it just states that they are amorphous pores .... I have also double membraned to make sure I wasn't blowing my protein through and thats not happening.
binding to pvdf can be influenced by sds. that's one of the reasons that methanol is included in the transfer buffer (to strip sds from the protein). if the transfer buffer contains more than 0.05% (or 0.1% depending on which source of information you read) then you may see inconsistent binding (it can also influence binding to nitrocellulose but nc also depends on electrostatic as well as hydrophobic interactions).
check out the troubleshooting section in this protein blotting handbook (6th edition) from millipore (emd merck), attached, and download the western blotting handbook from ge healthcare lifesciences from this webpage.