smearing of proteins during transfer - (Apr/10/2008 )
For the past four months I've been doing western blots using Tris-tricine and PVDF membranes to visualize a small protein. Initially I was doing wet transfers and everything worked well for about a month - I got good transfers using a standard glycine buffer/20% methanol. Then I stopped doing the experiments for a while and when I came back to it a month ago, I started to have problems with the transfer step. It looks like the proteins are smearing, sort of in a swirling pattern - as if the proteins are floating in the buffer for a while before they hit the membrane. Essentially, I don't think that the gel is making good contact with the membrane. I tried remaking the acrylamide, didn't help. I tried a number of different transfer buffers - didn't help. I then tried to do semi-dry transfer instead of wet transfer, and I'm getting ok results now (no smearing). However, when I reviewed all of my experiments over the past few months, I think that I was getting better results initially with the wet transfer than with the semi-dry transfer, so I would really like to go back and figure out what went wrong. Yesterday I remade all of the reagents (acrylamide, 3x buffer, etc) and followed the protocol exactly as I had initially done 4 months ago, but still I am getting the smearing. I can't figure out what is different. Other members of the lab use the same equipment and they are not having problems, so I don't think it’s an issue with the equipment (although I am the only one using PVDF membranes and Tris/tricine). Has anyone had this happen to them before? Any suggestions would be appreciated.
thanks,
smu
We had this problem once and we cleaned all the western blot stuff (instruments) like chambers and trays very well and the problem ceased. I am not sure if the cleaning helped or something else changed.
good Luck !!!
good Luck !!!
Will try this, thanks.
Two things to consider further if you haven't figured this out yet...
One, make sure your transfer chamber contacts/leads are still intact and are cleaned (try rinsing in many changes of dI H2O)
Two, make sure the transfer buffer is fresh each time you use it, including the methanol. If the methanol is older than 6-8 months old, replace it. I am betting the bottle is pretty big and pretty old too.
Hope this helps, Good Luck!
Rocky...
One, make sure your transfer chamber contacts/leads are still intact and are cleaned (try rinsing in many changes of dI H2O)
Two, make sure the transfer buffer is fresh each time you use it, including the methanol. If the methanol is older than 6-8 months old, replace it. I am betting the bottle is pretty big and pretty old too.
Hope this helps, Good Luck!
Rocky...
Thanks for taking the time to reply, but I don't think its either of those things. I can get the transfer to work fine on normal western blots using Tris/glycine (instead of Tris/tricine) and nitrocellulose - just did one today and it looks perfect. So the equipment seems to be fine. As for the methanol, several people are doing really big gels in our lab that require a lot of methanol to transfer, so our methanol is almost always less than two weeks old. If you think of anything else, let me know.
smu
Swirling is usually caused by poor contact between the gel and membrane. Make sure to pre-wet PVDF in Methanol, make sure the gel is wet when you put the PVDF on top of it, and make sure your sponges are still thick enough to hold everything tightly. Ours compress after a while and I either have to get new ones or use an extra. I hope this helps!
Our sponges are rather old and getting thin - would it help to use more whatman paper? Currently for wet transfers I use one sheet on front and back.
Hmm, at least in my wet transfer system (Invitrogen) adding an extra sheet of paper probably wouldn't add enough thickness, plus the paper isn't as spongy and you don't want too much pressure on the gel. Can you borrow some sponges from a neighbor? I don't think they are very expensive so it might be worth it to just replace them.
So I tried adding an extra sponge during the transfer and it seems to help a lot. I'm not getting the huge smearing problems that I was before. Now if I could just get these goofy antibodies to work. Thank you for your help!!!!!
Out of interest, why are you using PVDF membranes for tris-tricine and nitrocellulose for tris-glycine?
if you know nitrocellulose always works why not just stick to that? It's what i always use with tricine gels.