My problem is that in theory, I have to make the first dimension (native) run and the second dimension (SDS-page) run on the same day, and then transfer on membrane still on the same day. Bu also it would be better to have freshly prepared samples, to maintain native conditions.
I just wanted to add a question here as I am planning to run a BN gel as well, do you the dializing before as mentioned in some protocols? or what protocol do you do and does it work for you?
cheers
Peter
Hi Peter, up until now I''ve run the BN gel without dializing (I think you refer to the JOVE protocol). I've added comassie G-250 directly to the sample, 1/5 % of the detergent used (digitonin in my case, preliminary experiments with DDM did not give good results for my proteins). If you do not overload the gel, the run is fine. Personally I do not understand why dializing the samples, unless you use high concentrations of detergents (more than 1%). but I'm still learning in this field.
Nelson, many thanks for the suggestion, but when you say 50% glycerol..in water? or other buffer? I was also thinking to store (after alkylation) in transfer buffer...met-oh should fix the protein without too much diffusion...
For the electrophoresis procedures you should start them at the same day but you can win some time by doing the tranfer on membranes overnicht in a tank blot. Use a temparature of 15 °C (below this, SDS will start to crystallize).
Hello Fulvio, I have run 2D gels like yours and try different way of storage. Finally, the best way was to store the gel strip without any buffer in a polypropyléne tube at -20°c (not polystyréne and others polycarbonate) just one night (like with IEF strip actually). To save time with your blotting, have you a semi-dry apparatus?
You can simply store the gel strip without any buffer in distilled water in a polypropylene tube at low temperature. Stained proteined will not diffuse.
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