how to dissolve Polyacrylamide gel? - (Feb/27/2012 )
Hi All,
Does some body know how to dissolve Polyacrylamide gel? I want to segregate a lipopolysaccharide molecule (relatively DNA/RNA/ Protein Free), which I can detect on SDS PAGE using silver staining. I want to slice the gel band and hopefully dissolve poly acrylamide gel, without destroying product. are there any commercial kits available ? or any home grown recipe ? I have tried goggling it a lot WITHOUT MUCH SUCCESS.
Any help is appreciated.
Many Thanks,
Aman Singh
you need to use trypsin. check out the links:
http://plaza.ufl.edu/johnaris/Protocols/Protein/ProteinInGelTrypsin.pdf
http://www.protocol-online.org/biology-forums-2/posts/9081.html
you will not cut a lipopolysaccharide with trypsin which is a protease;
cut out the gel piece, choose appropriate pore size of dialyze tube, put gel piece into it, and run current in a horizontal gel chamber...
Something you could look into would be labile cross-linkers for your gel solution, they come in different varieties - acid labile, base labile, photolabile, enzymatic labile, etc. You can subject the gel to specific conditions and cause it to dissolve.
if you don't have labile crosslinker, as suggested by proteamatt, then you can digest polyacrylamide with the wet oxidation method:
using freshly prepared 60% perchloric acid and 30% hydrogen peroxide,
slice the gel into 3-5mm strips with a sharp blade. place the slices into a glass vial (we use scintillation vials), one per vial.
add 0.8ml of 30% peroxide in a slow, dropwise fashion.
add 0.4ml 60% perchloric acid in a dropwise manner (you can use 0.4 and 0.2ml, respectively).
seal the vials with teflon or polyethylene lined caps (do not use foil-lined caps).
incubate in oven at 70C until gel slice is digested (3 hours is typical digestion time).
cool and adjust the pH to 6-7 with 5N sodium hydroxide (~0.4ml), check pH with pH paper).
mdfenko on Wed Feb 29 20:17:09 2012 said:
if you don't have labile crosslinker, as suggested by proteamatt, then you can digest polyacrylamide with the wet oxidation method:
using freshly prepared 60% perchloric acid and 30% hydrogen peroxide,
slice the gel into 3-5mm strips with a sharp blade. place the slices into a glass vial (we use scintillation vials), one per vial.
add 0.8ml of 30% peroxide in a slow, dropwise fashion.
add 0.4ml 60% perchloric acid in a dropwise manner (you can use 0.4 and 0.2ml, respectively).
seal the vials with teflon or polyethylene lined caps (do not use foil-lined caps).
incubate in oven at 70C until gel slice is digested (3 hours is typical digestion time).
cool and adjust the pH to 6-7 with 5N sodium hydroxide (~0.4ml), check pH with pH paper).
Thats pretty cool. I learn something new everyday
not so new, i learned the method 35-40 years ago.
Does this treatment leave anything behind after digesting the polyacrylamide? It sounds ridiculously harsh for isolating biomolecules.
Also, if anyone thinks about doing this, make sure to read about the explosion hazards of perchloric acid.
it takes harsh conditions to digest polyacrylamide. we used it to prepare radioactive samples for scintillation counting, so it didn't matter if the molecule wasn't completely intact.
if i want to extract intact proteins from a gel then i would electroelute or homogenize the gel in buffer and collect the liquid.
i figure that, since the gel was sds-page and silver stained, amansingh was not trying to isolate biochemically viable protein.