I am following one protocol by that I am trying to take out nucleus proteins but one of my buffer having SDS and in this case I can not use the same sample for 2DE analysis. I tried another detergent on place of SDS but in that case I was not able to dissolve the chromatin protein pellet.
If any of you knows how to remove SDS from protein sample for 2DE analysis, kindly share you view or experience on research gate.
I would like to give further information if neede.
Hi Amit, there are two ways of removing SDS from your protein sample.
First is to precipitate your sample with organic solvents such as acetone; this is simple, inexpensive, and works very well. Add 5 volume of pre-chilled acetone and incubate in a freezer for >30 min. Then spin at max rpm for 10 min, discard the supernatant, wash the pellet with 75% acetone, and dissolve the pellet in your buffer of interest. The resulting protein pellet is often hard to completely solubilize; in this case, supplement your buffer with some chaotrophic agent (e.g. 8 M urea, or 6 M urea plus 2 M thiourea).
Alternatively, you can use size cut-off membrane filters. See the attached for details.
Thanks Dooyoung lee and pavel actually I was aware about some of the protein precipitation methods but was not sure that it will remove the SDS but after reading a informative discussion I use one of the discussed method for removal of SDS.
I will also try to use Amicon if it works fine the I would like to with this.
Thank you so much both of you
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