Originally Posted by mai-mb
Yeah after a run that long I would expect the transfer buffer to be used up,
I use very little simply saturating the buffer sandwich, but after the hour 47mins the sandwich is still quite saturated.
...... do you heat your samples at 95 or 100?
WOOW!!, hold it, my sandwiches are bone dry after semi-dry blotting 19-20min as described before. So that is your problem, this is not normal.
You probably have bad contact, perhaps the blot filter papers are not the right thickness, double/triple up and problem solved. Or maybe the little springs in the semi-dry blot apparatus are very compressed and not pushing the platinum anode out enough---just push them out for them to decompress, slowly and gently.
[Could also be buffer just isn't running buffer, no ions to run---check that the buffer makeup is actual running buffer recipe and concentration (proper water source was used).]
Clean the semi-dry apparatus (not the power source) with copious amount of DI water make sure buffer salts aren't beneath the plaforms (both lid and bottom), terminals (where power source wires make contact)---then leave them Upside Down over paper towels overnight to drip to complete dryness. Check for a springiness to the apparatus lid, the platinum coated platform should be bouncy, any flaky stuff and you have salt buffer build-up in there.
Yes samples are boiled in Laemmli loading buffer for 5 minutes, then quickly centrifuged before loading on gel.
Remember after the blotting, you can coomassie or Imperial Blue stain the gel to check transfer efficiency.