Roles of staining soluton components!!! - (Jul/21/2010 )

Hey people!!! may seem to be a very trivial question but i m really confused!! may be a bad day at science!!!
this happens wen u work the next day after ur engagement!!!!

Can anyone tell me the exact roles of acetic acid/methanol in fixing solution?
Why an IEF gel is prefered to be fixed in 10% TCA and not the above mentioned fixing solutions?
What are the roles of phosphoric acid and methanol in colloidal staining solution?

acetic acid fixes the protein, methanol prevents the gel from swelling (and may cause it to shrink). with coomassie stain the methanol also helps solubilize the dye.

many ief gels are polymerized onto a plastic support. methanol is not necessary to prevent swelling. tca is a better protein precipitant (fixative) than acetic acid.

colloidal stain uses ammonium sulfate to form the dye colloid. methanol breaks up the colloid and solubilizes the dye so it is not used or is kept at a low concentration (used to initially solubilize the dye) to avoid breaking the colloid.

phosphoric acid is used in the coomassie protein determination (bradford), i am not aware of its presence in the colloidal dye solution (that doesn't mean it won't be there in at least one formulation of the colloidal stain).

thanx md... yet again!!!!

is tere a reason then to not use 10% TCA for fixing SDS-PAGE gels!!!

in bradford i think acid is tere cause the reaction proceeds only in acidic conditions... may be acidic pH is necesaarry so the phosphoric acid!!! dunno if it can be substituted by any other acid though!!!!

Prep! on Jul 21 2010, 11:43 PM said:

the gel will swell but you can use it (you may require extensive washing afterwards, depending on staining technique).

the phosphoric acid enhances the color shift.

yeah may be any other acid wont be as effective as phosphoric acid!!
i also always wonder wat is the role of sulphosalicyclic acid in fixing IEF gels along with TCA??!!!! i tried with and withough it but its making no difference to me!!!

Prep! on Jul 23 2010, 01:35 AM said:

i'm not sure either. it was used in older protocols. the newer protocols just call for 20% tca.

i just looked it up... sulfosalicylic acid is a protein precipitant.

mdfenko on Jul 24 2010, 12:57 AM said:

precicely... its the same purpose as TCA... still it s used!!!!