Gel-elution

Hiranya Roychowdhury hroychow at NMSU.EDU
Wed Nov 29 12:26:18 EST 1995


It is also possible to elute proteins from gel strips by reverse 
electrophoresis:

Polymererize 15% gel, filling half of the sandwich. 

Place the gel pieces that contain the protein(s) of interest on the 
polymerized gel. These are inserted into the sandwich with the help of a 
spacer or a spatula, so that the strips sit evenly on the 15% gel surface.

Next, fill up the remainder of the sandwich with stacking gel, leaving 
about a couple of centimeters from the top empty. let it polymerize.

Run the gel with the usual buffer system, BUT in reverse. See the dye 
migrate upwards. If the original gel was not stained, stain the gel 
slices lightly with BPB prior to loading them.

As the stain reaches the top of the stack, overlay the surface with 5-10 
mL (depending on the thickness of the gel sandwich) of 1x Laemmli sample 
buffer (w/ 10% glycerol) and collect the eluent every 10 min with a 
syringe fitted with a 18 or 20 g needle. It is a good idea to put a 
teflon tube sleeve on the needle to avoid damaging the gel surface. The 
eluent is then monitored in a spec (blank with the same buffer) at 280 nm.

This method works fine and does not need separate equipment for elution. 
It also recovers, depending on the experience of the worker, about 90% of 
the protein from SDS-PAGE slices if the gel was not fixed and stained 
with acetic acid. Cu++ stained gels are the alternative where recovery 
approaches 100%.


			>>>>>>>>>>>>>>>>>>>>>>>>>>>
			  Hiranya S. Roychowdhury
   			  Plant Genetic Engineering Lab.
			  Box 3GL, NM State Univ.
			  Las Cruces, NM 88003
			  Phone: (505) 646-5785
			  hroychow at nmsu.edu
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