Editing SDS-PAGE Gel Staining (section)

== Protocol 2 ==



Blue Silver staining method uses Coomassie Brilliant Blue G250 for the staining reaches sensitivity upto 1ng. The method was originally developed by Neuhoff and further modified by Righetti et al, 2004.

Procedure<br>1) Following the electrophoresis fix the resolved protein with Acetic acid: Methanol: Water 1:3:6. Alternatively One can use 10% Phosphoric acid.<br>2) To the CBB G250 solution add 20% Methanol just prior to use with constant stirring to allow the formation of colloid effectively.<br>3) Add the above prepared solution to the gel and keep on a gel rocker with constant shaking.<br>4) Protein bands take up bright blue stain while the background remains faintly stained with this procedure.<br>5) Briefly rinse the gel with Tris-Phosphate solution pH6.8.<br>6) Stabilize the protein-CBB G250 complex with 20% ammonium sulphate solution for 30min.<br>7) Rinse with water and store the gel indefinitely in distilled water.

<br>Recipes<br>CBB G250 solution - 0.12% CBB G250 in 10% Phosphoric acid, 10% ammonium suphate and 20% methanol.<br>The CBB G250 solution is prepared as follows:<br>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<br>solids have dissolved, add water to 80% of the final volume. To this solution, under stirring, add methanol to reach a 20% final concentration. This stock dye solution should be kept in a brown bottle and is stable at room temperature for almost 6 months.<br>

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