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Which wash buffer (to collect cell) is prefered for 2DE?


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#1 yimao

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Posted 20 March 2011 - 12:05 AM

PBS or the one with Tris + Sucrose? thx~

btw: any good method to store the SDS gel after silver stain? i mean for a longer period of time, like months?

#2 mdfenko

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Posted 21 March 2011 - 08:09 AM

tris+sucrose has lower ionic strength (presumably, since you didn't give any concentration information) so will have less effect on electrophoresis.

if we don't dry them, we store our gels in coomassie destaining solution (30%methanol, 7% acetic acid). the balance of methanol and acetic acid maintain the physical dimensions over the long term (as long as you occasionally replace the solution).
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#3 yimao

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Posted 21 March 2011 - 07:20 PM

tris+sucrose has lower ionic strength (presumably, since you didn't give any concentration information) so will have less effect on electrophoresis.

if we don't dry them, we store our gels in coomassie destaining solution (30%methanol, 7% acetic acid). the balance of methanol and acetic acid maintain the physical dimensions over the long term (as long as you occasionally replace the solution).


thx, man.

i use 0.6g Tris, 42.8g Sucrose dissolve in 500ml h2o, then adjust the ph to 7. (10mM Tris, 250mM Sucrose, pH 7.0)

could coomassie destaining solution also preserve the protein in gel as long too? kinda need to do some MS protein identification after that.

#4 mdfenko

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Posted 22 March 2011 - 07:37 AM

could coomassie destaining solution also preserve the protein in gel as long too? kinda need to do some MS protein identification after that.

the acetic acid denatures and maintains the protein in the gel, although, with silver stains you are treating with formaldehyde which will also lock the protein into the gel matrix.

most silver stain techniques are not compatible with ms. ensure that the one you use is (there are some kits that are for ms after staining like this one from thermo-pierce).
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