Editing SDS-PAGE Gel Staining (section)

== Blue Silver&nbsp;: most sensitive method ==

10% phosphoric acid

10% ammonium sulfate

0.12% G-250

20% methanol or EtOH




How to make:

make a solution containing 10% phosphoric acid,&nbsp; 10% ammonium sulfate, 0.12% G-250. Then while stir slowly, slowly (drop by drop) add methanol

To 100 ml water add phosphoric acid (enough to obtain 10% in the final 1L).

*Add 100 g ammonium sulphate.
*Add 1.2g Coomassie Blue G-250.
*Add 116 mL of 85% o-phosphoric acid
*Add water to 800 mL.

Dissolve by stiring for 1 hour. then:

<br/>• Very slowly using peristaltic pump or flow rate adjusted column: add 200 mL of 100% methanol.<br/>



The critical point lays in the mode of addition of methanol, which facilitates the staining. Methanol addition to the solution of 10% phosphoric acid, and 10% ammonium sulfate is a critical step. It should be added extremely slowly (ideally in a 'drop-by-drop' manner, i.e., in small amounts and not in one step), under continuous and vigorous stirring in order to preserve the micellar colloidal suspension of the dye. This improves sensitivity. Indeed, the final physicochemical conditions of the solution are not the same in either case. The physical state of the solution can be easily inferred by the color of the solution: it will be a dusty greenish-blue when addition has been performed slowly but light blue if addition has been carried out incorrectly (too quickly). In the latter case, the homogenous blue color denotes dispersion of the micelles, with loss of the improved sensitivity. When addition of methanol is done in a one-step procedure instead of adding it 'drop by drop' as stated above with extremely gentle stirring (being perhaps the only time-consuming step, to be done the day before need) colloidal micelles are suddenly dispersed and the color of the solution shifts from a greenish dusty deep blue to a brilliant homogenous blue (typical of the formation of unprotonated anionic species, and much less efficient in complexation with proteins). When micelles are dispersed, the sensitivity of the method drops from 1–5 ng down to 10 ng or worse. The state of the micelles is pivotal, therefore any care must be taken in avoiding micelle solubilization

<br/>Sensitive staining:

1. Fix gel in 50% ethanol and 2% phosphoric acid.<br/>2. Wash the gel for 2x 20 minutes in ddH2O.<br/>3. Add staining solution and stain overnight or longer.<br/>4. Rinse the gel with ddH2O and destain with ddH2O using microwave.<br/><br/><br/>Destaining: water

rinsing and fixation in 20% methanol