# Gel Staining Procedure for PAGE > 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... Canonical URL: https://biologynotesonline.com/gel-staining-procedure-for-page/ Author: Sourav Pan Last updated: April 16, 2024 ![Gel Staining Procedure for PAGE](https://biologynotesonline.com/wp-content/uploads/2022/01/Gel-Staining-Procedure-scaled-1.jpg) ## 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 - 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. - 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. - After staining Wash your gel several times using distillate water to eliminate any stain. - 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. - After destaining, gels can be stored in gel storage solutions and photographed when needed. ![Coomassie Blue staining ](https://biologynotesonline.com/wp-content/uploads/2024/04/image-2079.png)Coomassie Blue staining ## 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 - After the electrophoretic run, immerse the gel in the fixer for 40 minutes. An overnight soak in fixer will create a more clear background. - 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. - Then, decant the water and soak the gel in the sensitizer solution for 10 minutes. - Clean the sensitizer solution off and wash the gel 2 times with water, each time lasting 10 minutes. - Discard the water and soak in the gel 10 minutes in the silver solution. - Dissolve silver in a solution and rinse the gel with water for 1.5 minutes. - Then, discard the water and then immerse the gel in the solution of developer for 3-7 minutes. - 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. - 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 - Submerge the gels following electrophoresis in the fixing solution 20 minutes. Repeat this process two times. - Rinse the gel in the water two times, each time lasting 30 minutes. - Incubate the gel in the staining solution for 20-40 minutes with gentle stirring. - Get rid of any stain using 10 percent Acetic acid. - 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 - Rinse the gels following electrophoresis in distillate water for a maximum of 30 minutes. - The gel should be immersed within a 0.3 M CuCl2 solution for 10 min. - Rinse the area with de-ionized, water. - Proteins can be visualized as clear areas on blue backgrounds. - 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. - Transfer the gel with the stained area to the transfer buffer prior to starting the transfer set-up.