Protocol Online logo
Top : New Forum Archives (2009-): : SDS-PAGE and Western Blotting

How to prepare colloidal coomassie blue? - ingredients can not dissolve (Jun/08/2009 )

Hello everyone,
I was trying to make colloidal coomassie blue stain following the protocol:

Electrophoresis 2004, 25, 13271333
The dye solution is prepared as follows, by sequentially adding the various ingredients as here indicated: to a water solution (1/10 of the final volume) the desired amount of phosphoric acid is added, so that, in the final volume, its concentration will be 10%; to this, add the required amount of ammonium sulfate (in powder), calculated to obtain a final concentration of 10%. When the ammonium sulfate has dissolved, add enough Coomassie Blue G-250 (in powder) to obtain a final concentration of 0.12%. When all solids have dissolved, add water to 80% of the final volume. To this solution, under stirring, add anhydrous methanol to reach a 20% final concentration.

However, ammonium sulfate and Coomassie Blue G-250 can not dissolve completely. Should it be stirred for longer time?

-WHR-

If ammonium sulfate and coomassie blue are first dissolved in water and methanol separately, than mix with phophoric acid, all the stuffs can disolve readily. I don't know the reason why they should be added in that order.

-WHR-

don't add the coomassie until all of the ammonium sulfate is in solution. if necessary you can add some more water to get all of the ammonium sulfate into solution.

the coomassie won't go into solution (it becomes a colloid), but, as long as the ammonium sulfate is completely in solution, you can stir it for a while until well dispersed then add the methanol.

-mdfenko-

mdfenko on Jun 8 2009, 08:20 AM said:

don't add the coomassie until all of the ammonium sulfate is in solution. if necessary you can add some more water to get all of the ammonium sulfate into solution.

the coomassie won't go into solution (it becomes a colloid), but, as long as the ammonium sulfate is completely in solution, you can stir it for a while until well dispersed then add the methanol.


Thank you for your advice.
This reagent really work fine. A 10 ng BSA band is visible after 1 hour fixation and 1 hour staining. The background was clean.

-WHR-

I join recently. although it is too late reply but still can help. you should dissolve coomassie and ammonium sulfate separately. allow more time to coomassie to dissolve .mix both solns and then add phosphoric acid. add methanol just before stain the gel .it will give best colloids.

-Tarum Irum-