protein fragment on SDS-PAGE less than 3 KDa.... - how to detect it? (Mar/27/2006 )
i want to establish SDS-PAGE where I can see together 58kDa protein and around 2KDa fragment....up till now i have only found Schagger's paper (1987) describing some SDS-PAGE manipulation....i just thought i ask for your experience here....thanx a lot
I have used the Schagger and von Jagow method for a few years and I find that if I need to separate in that range then I use a 10- 20% gradient with a 4% stack. I omit the 10% spacer when I run a gradient, I otherwise follow their formulation.
Run at 50V for 20-30 minutes then 90-100V for 16 hours for a full size gel.
45-50V for 15 min then 110V for 1.5-2 hours for a mini gel.
stain as usual.
We had tried a few other methods to separate low mw proteins and found this to be the best for our purposes.
thank you very much, do I need a special equipment since i have to seperate anode and cathode buffers? i dont think its possible with the one im using right now....is there any trick? 
what kind of equipement do you have?
Usually you put buffer between the two gels. It's in contact with the upper part of the gel, it's where you put the cathode buffer
You also put buffer in the tank. It's in contact with the lower part of the gel, it's where you put the anode buffer.
I usually use 12% separating gel with 4% stackting gel under 100 volts approximately 2.5-3 hours.
Hope it work for you !
most of the gel boxes have separate anode and cathode tanks (many have removable upper tanks). just add the lower buffer first then carefully add the upper buffer, trying not to spill any into the lower tank.
thank you everyone!
what kind of equipement do you have?Usually you put buffer between the two gels. It's in contact with the upper part of the gel, it's where you put the cathode buffer
You also put buffer in the tank. It's in contact with the lower part of the gel, it's where you put the anode buffer.
Missele, you are right.....but here we put between the gels the buffer that is "new running buffer" and to the lower tank "used running buffer"...and after each run all the buffers are mixed together and we empty them into the "used running buffer "bottle to use it again in the lower part....that is why i am confused, thinking that both buffers might get mixed during the run...
just throw out the buffers after the run.
Kathy- another trick if you are worried about that little guy blowing through during transfer, put two membranes back to back in the transfer apparatus...perhaps the second one will catch it if it goes through