We use the stainfree system from bio-rad. We use 4-15% TGX criterion stainfree gels and have recently observed membranes with fussy band safter transfer, pre-stained standards which have "ran" out as well as samples smeared out. After running the gel we made stainfree pictures of the gel, and the gels looked all fine. The problems we have observed are after the transfer. We use bio-rad turbo transfer, set at 2.5amp 25V 7 min (premade protocol from bio-rad).
It actually sometimes looks as if the gel has melted, and we have run samples 6 - 12 month ago with good results and now the same samples suddenly look bad. We do not prepare membranes for transfer ourselfes, we use readymade turbo transfer packs PVDF membranes also from bio rad, and have done so for 2 years with very good results. Has anyone here experienced anything like this?
Now we use wet transfer, using the same gels, preparing our own membranes (PVDF) and it looks very nice.
We are discussing if it could be the buffer in the ready made transfer pack, the membrane the turbo transfer mashine or perhaps the gels.
The two files reprecent the gel and membrane from the same experiment, stainfree pictures taken with a chemidoc camera ( bio rad ).
As you mentioned, it might be due to increased gel temperature/meting during transfer. Check the buffer prepared. Also check the trouble shooting section of the transfer module specific for the instrument.
A potential problem may be that the contact between the gel and transfer pack is weak leading to potential bubbles/dry areas (which will cause that odd smearing pattern at the low end). Just make sure you roll evenly and carefully and expedite the time it takes to run it in the turbo. Hopefully that helps!
I would also check the expiration date on your gels if they are ready made. I have had old ones work fine for the electrophoresis and disintegrate during transfer. Did the gels look melted at all after the transfer?
Have you tried to run a second gel in parallel and stain with blue Coomassie for proteins? Your problem might be the gel . I personally always transfer in wet condition although if the sandwich ( gel, filter, membrane) is moist enough, and there are no air bubbles , blotting in semi-dry shouldn´t be a problem.
thank you for the answers. We do not stain the gel, we use the stainfree system from bio rad, we can see the proteins in the gel rigth after it has been run and also the membrane after transfer. The gels has always run nicely, the smeared bands and prestained standards only appear on the membrane, therefore we belive it is the transfer. We have now run severel gels using wet transfer with no problems. The gels we use are not "out of date" The gels also do not look melted, only the protein.
it could be the APS I used fresh not freezed, the acrylamide sollution should be not more than one month
It happened to me few weeks ago. Placing the biorad's "sandwich" I noticed that it wasn't set as usually. There was a bump so I tried with a roller to get rid of it and I thought I succeeded until I saw the standards all blury. Revealing my proteins I saw something like your second image. So I think it was because of that bump. It can be difficult to seize all the layers without a bump. i don't know if you noticed that kind of stuff.
Hope it helps
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