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how to dissolve Polyacrylamide gel?

Polyacrylamide Gel Lipopolysaccarides lipopolysaccharide

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8 replies to this topic

#1 amansingh

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Posted 27 February 2012 - 02:50 PM

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

#2 Curtis

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Posted 28 February 2012 - 02:04 AM

you need to use trypsin. check out the links:

http://plaza.ufl.edu...nGelTrypsin.pdf

http://www.protocol-...posts/9081.html

#3 Inmost sun

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Posted 28 February 2012 - 03:26 AM

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...

#4 proteaMatt

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Posted 28 February 2012 - 07:27 AM

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.
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#5 mdfenko

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Posted 29 February 2012 - 12:17 PM

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).
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#6 proteaMatt

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Posted 29 February 2012 - 12:28 PM

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 Posted Image
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#7 mdfenko

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Posted 29 February 2012 - 12:41 PM

not so new, i learned the method 35-40 years ago.
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#8 phage434

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Posted 01 March 2012 - 05:51 AM

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.

#9 mdfenko

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Posted 01 March 2012 - 11:44 AM

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.

Edited by mdfenko, 01 March 2012 - 11:44 AM.

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Also tagged with one or more of these keywords: Polyacrylamide Gel, Lipopolysaccarides, lipopolysaccharide

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