SDS PAGE Destaining - (Jan/21/2015 )
Does anyone ever tried to destain a SDS PAGE gel with a home made solution of acetic acid and methanol using, vinegar and burning alcohol?
I have trouble buying this chemicals because of the drug regulation of my country...
Any other ideas in how to do this?
Note: I don't have the CBB G to do the colloidal Coomasie. I have only Brillant Blue R
for alcohol, you can use wood or grain alcohol (methanol or ethanol).
your problem is the vinegar. white (distilled) vinegar is usually about 5% acetic acid. most vinegars will not exceed ~12%.
destaining solution requires at least 5% acetic acid (final concentration). we use 7%. you will not be able to make these concentrations with household strength vinegar.
and, acetic acid is required to ensure that the proteins remain fixed in the gel (as well as controlling the shrinkage caused by the methanol).
a possible way around this would be to prefix your proteins in the gel with tca (probably better to use a mixture of tca and sulfosalicylic acid). if you do this then you have to ensure that you wash out residual fixative prior to staining or the stain will precipitate. then you will be able to destain with an alcohol solution without having to worry (as much) about washing out your protein.
mdfenko on Fri Jan 23 13:36:29 2015 said:
for alcohol, you can use wood or grain alcohol (methanol or ethanol).
your problem is the vinegar. white (distilled) vinegar is usually about 5% acetic acid. most vinegars will not exceed ~12%.
destaining solution requires at least 5% acetic acid (final concentration). we use 7%. you will not be able to make these concentrations with household strength vinegar.
and, acetic acid is required to ensure that the proteins remain fixed in the gel (as well as controlling the shrinkage caused by the methanol).
a possible way around this would be to prefix your proteins in the gel with tca (probably better to use a mixture of tca and sulfosalicylic acid). if you do this then you have to ensure that you wash out residual fixative prior to staining or the stain will precipitate. then you will be able to destain with an alcohol solution without having to worry (as much) about washing out your protein.
Thanks mdfenko
I will try this!!
Mag 22388 on Fri Jan 23 17:33:44 2015 said:
mdfenko on Fri Jan 23 13:36:29 2015 said:
for alcohol, you can use wood or grain alcohol (methanol or ethanol).
your problem is the vinegar. white (distilled) vinegar is usually about 5% acetic acid. most vinegars will not exceed ~12%.
destaining solution requires at least 5% acetic acid (final concentration). we use 7%. you will not be able to make these concentrations with household strength vinegar.
and, acetic acid is required to ensure that the proteins remain fixed in the gel (as well as controlling the shrinkage caused by the methanol).
a possible way around this would be to prefix your proteins in the gel with tca (probably better to use a mixture of tca and sulfosalicylic acid). if you do this then you have to ensure that you wash out residual fixative prior to staining or the stain will precipitate. then you will be able to destain with an alcohol solution without having to worry (as much) about washing out your protein.
Thanks mdfenko
I will try this!!
Do you think that I could do a fixation using just the vinegar first and then destaining wiht the alcohol?
Mag 22388 on Fri Jan 23 17:36:12 2015 said:
Do you think that I could do a fixation using just the vinegar first and then destaining with the alcohol?
no. fixation with acetic acid is reversible.
I totally missed the alternative ingredients part - How about adjusting water amount such that final acetic acid conc is 5%? You may have to use 850 ml of vinegar if it is about 5% acetic acid.
CPRES on Fri Jan 23 23:39:21 2015 said:
I totally missed the alternative ingredients part - How about adjusting water amount such that final acetic acid conc is 5%? You may have to use 850 ml of vinegar if it is about 5% acetic acid.
15% alcohol may not work too well when destaining (although some may use 10% ethanol, we use this to remove sds when silver staining, never tried to destain with this). we routinely use 30% methanol. the only time we reduced alcohol in the destaining solution was when we destained electrophoretically (we used cylindrical gels back then), then we used 5% methanol, 7.5% acetic acid.