PAGE staining

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PAGE Staining Methods

Coomassie blue (G-250) staining:

Sensitivity: 0.2~0.5μg/band

  • Staining solution:

0.3 % Coomassie Brilliant Blue R-250 (w/v)

45 % Methanol (v/v)

10 % Glacial acetic acid (v/v)

45 % dwater

 

  • Destaining solution:

20%Methanol (v/v)

10% Glacial acetic acid (v/v)

70% dwater

 

  • Procedure:

1. Immerse the gel with staining solution,and slowly shake it on horizontal rotator for about 20-30min.

You can microwave it to shorten the staining time to 1-3 min, but the microwave time must not exceed 10 seconds to avoid boiling.

2. Immerse the gel in destaining solution and put it on the same shaker for about 20-30min.

Change the destaining solution for 3-5 times untill you can see clear bands with almost no blue background.

You can also microwave it and watch the time.

Silver staining:

Sensitivity: 2ng/band

Fix and develop. Disadvantages: complicated procedures and high cost.

1-    100ml fixing solution : 50 % Methanol, 10% Acidic Acid, 50 µl Formalaldehyde

for one hour to overnight.

2- Wash gel with 50 % ethanol for three times (no less than 20 minutes each).

3- Treat gel for 1min with hypo solution (Sodium Thiosulfate

solution 20 mg/100 mL). SAVE 2 ml FOR LATER STEP.

Over treating with the hypo solution will result in darker gel in the end.

5- Wash with water for three times (20 seconds each).

6- Treat gel with Silver Nitrite solution (200 mg/100 mL) for 30 minutes.

Over treating will result in unwanted artifacts in final stage.

7- Wash with water for three times (20 seconds each).

8- Develop gel in 100 mL developing solution (6 g Sodium Carbonate, 2 mL of the hypo solution, 50 µl Formalaldehyde). This step can take from one minute to 30 minutes, depending on protein concentrations. If you overdevelop, gel will turn dark brown

9- Once developed, stop with 5% Acidic Acid. Once you see the bands, stop developing the gel immediately.

10- Store gel in fixing solution. Do not store for too long, try to dry the gel as soon as possible.

Fluorescent dye staining:

Sensitivity: 5ng/band

No background, but more difficult to capture the result