Gel Staining Procedure for PAGE

A. Coomassie Blue staining

Staining protein gels using Coomassie Brilliant Blue R-250 is common to see proteins that are resolved using SDS-PAGE. It is extremely sensitive and suitable for long-term storage of gels.

Reagents Required

• Gel Fix solution (500 mL) [Methanol (M3641) 250 mL, Glacial acetic acid (695092) 50 mL, Water 200 mL]
• Coomassie solution [CBB R-250 (B6529) 0.1%, Methanol (M3641) 40%, Glacial acetic acid (695092) 10%, Filter the stain solution using Whatmann No. 1 filter paper.]
• Destain solution [Methanol (M3641) 50 mL, Glacial acetic acid (#695092) 35 mL]
• Gel storage solution [Glacial acetic acid (695092) 25 mL, Water 475 mL]

Procedure

1. After electrophoresis is completed, place the gel into the tray made of plastic that contains Gel Fix Solution. Set the tray on a rolling table and set the proteins for two hours.
2. Take the gel fix solution off and then add Coomassie solution. Set the table on a rocking surface and leave the gel staining for 2 to 4 hours.
3. After staining Wash your gel several times using distillate water to eliminate any stain.
4. Add the destaining solution to the gel. Place it on the table to wait for approximately 4 hours, until blue bands with a clean background are evident.
5. After destaining, gels can be stored in gel storage solutions and photographed when needed.

B. Silver Staining

We have a very sensitive silver stain detection kit for gels using SDS-PAGE.

Reagents Required

The following reagents are additionally needed:

• Fixing solution [Ethanol (E7023) 50 mL, Acetic acid 10 mL, Water 40 mL]
• 30% ethanol (E7023) solution

Procedure

1. After the electrophoretic run, immerse the gel in the fixer for 40 minutes. An overnight soak in fixer will create a more clear background.
2. Remove the fixing solution , and clean it off for 10 minutes with a 30% solution of ethanol and then rinse with ultrapure water for 10 minutes.
3. Then, decant the water and soak the gel in the sensitizer solution for 10 minutes.
4. Clean the sensitizer solution off and wash the gel 2 times with water, each time lasting 10 minutes.
5. Discard the water and soak in the gel 10 minutes in the silver solution.
6. Dissolve silver in a solution and rinse the gel with water for 1.5 minutes.
7. Then, discard the water and then immerse the gel in the solution of developer for 3-7 minutes.
8. Add 5 mL stopping solution in the solution for the developer, and allow to incubate for 5 minutes. Remove the stop solution/developer and rinse the gel with ultrapure water for about 15 minutes.
9. The gel is able to be photographed and then kept in clean, ultrapure water.

To double stain you can stain the gel with CBB R-250 and then silver stain using the methods in the previous paragraphs.

The Fluorescent Stains are: We provide the fluorescent SYPRO and Lucy staining for electrophoresis of proteins. The gels can be submerged in the stain’s fluorescent light in dark , or the gel could mix with buffer cathode in the electrophoresis.

Reversible Gel Staining

Reversible gel staining allows the user to go on to the western-blotting process of protein immediately following SDS-PAGE.

A. R-PROB staining

R-PROB is an exclusive stain that detects proteins on gels as well as western blots.

Reagents Required

• Reversible Protein Detection Kit for Membranes and Polyacrylamide Gels (RPROB)
• Fixing solution (F7264)
• 10% acetic acid
• EDTA 50 mM

Procedure

1. Submerge the gels following electrophoresis in the fixing solution 20 minutes. Repeat this process two times.
2. Rinse the gel in the water two times, each time lasting 30 minutes.
3. Incubate the gel in the staining solution for 20-40 minutes with gentle stirring.
4. Get rid of any stain using 10 percent Acetic acid.
5. To remove staining remove the gel, clean it with EDTA then follow by two washes lasting 15 minutes each in either water or a fixing solution.

B. Copper staining

To verify the movement as well as separation of protein the gel can be stained using the use of a reversible stain like CuCl2.

Reagents Required

• 0.3 M CuCl2 solution
• 0.25 M Tris and 0.25 M EDTA solution

Procedure

1. Rinse the gels following electrophoresis in distillate water for a maximum of 30 minutes.
2. The gel should be immersed within a 0.3 M CuCl2 solution for 10 min.
3. Rinse the area with de-ionized, water.
4. Proteins can be visualized as clear areas on blue backgrounds.
5. To remove the stain completely to make a western blot clean the stained gels with 0.25 M Tris solution and 0.25 M EDTA solution, pH 9, repeatedly.
6. Transfer the gel with the stained area to the transfer buffer prior to starting the transfer set-up.