PROTEIN STAINING
 
Last Update: December 2006
 
 
PREPARE SOLUTIONS
1. 10X Ponceau solution (100 mL):
Mix 2 g of Ponceau-S (2%), 30 g of Trichloroacetic acid (30%), 30 g of Sulfosalicylic acid (30%), and dH2O to 100 mL
2. Silver Stain Wash Solution 1 (100 mL):
Mix 40 mL of Ethanol, 10 mL of Acetic Acid, and 50 mL of dH2O
3. Silver Stain Wash Solution 2 (100 mL):
Mix 10 mL of Ethanol, 5 mL of Acetic Acid, and 85 mL of dH2O
4. Silver Stain 5% Glutardialdehyde/5mM DTT (100 mL):
Mix 5 mL of Glutardialdehyde, 0.077 g of Dithiothreitol (DTT), and add dH2O to 100 mL
5. 0.6% Silver Nitrate (100 mL):
Mix 0.62 g of Silver Nitrate with 100 mL of dH2O
6. Na2CO3 Stock solution (Store at room temperature) (1 L):
Mix 25 g of Na2CO3 in 1 L of dH2O
7. Silver Stain Developer (Make fresh for day of use) (250 mL):
Mix 200 mL of Na2CO3 Stock solution with 50 mL of Formaldehyde
8. Silver Stain Stop Solution (1 L):
Mix 37 g of Tris-HCl, 25 g of Sodium Thiosulfate, and 1 L of dH2O
9. Silver Stain Preserving solution (1 L):
Mix 70 mL of Glycerol, 100 mL of Ethanol, and 830 mL of dH2O
10. Coomassie Blue (500 mL):
Mix 0.5 g of Coomassie Brilliant Blue with 500 mL of Destain solution
11. Destain Solution (1 L):
Mix 300 mL of Methanol, 100 mL of Acetic Acid, and 600 mL of dH2O

12. Preserving Solution (500 mL):
Mix 25 mL of Glycerol, 50 mL of Acetic Acid, and 425 mL of dH2O
   
NOTE: each staining technique has it pros and cons. Make sure you know exactly what you need before working with one of them
   
Ponceau staining
   
PROCEDURE
This is a reversible, sensitive staining of proteins
1. Dilute 10X Ponceau solution to 1X and incubate membrane for 5-20 minutes while rocking

2. Remove stain (safe) and destain excess with water (optional: buffers such as PBS and TBS remove the stain much faster). Do this with 2-5 changes. Protein positions should be obvious within 5 minutes of destaining (>2 changes of solution)
OPTIONAL: membranes can be stored in this state
3. Mark any positions with an appropiate marker (colored pencils work very well for this) and continue destaning until all the stain has been removed
NOTE: this staining can be performed at any time, even after a western blot has been performed and the positions of the proteins needs to be determined
   
Silver Staining
   
This is a permanent, very sensitive staining of proteins

All incubations are at room temperature
1. After running the gel, incubate it in dH2O for 5 mins. Follow normal fixation step with 30% Methanol:10% Acetic acid for 1 hour
2. Incubate in Wash solution 1. Repeat for a total of 3 times, 15 minutes each wash
3. Incubate with Wash solution 2 overnight

4. Wash with dH2O 2 times, 5-10 mins each wash

5. Incubate with 5% Glutardialdehyde/5mM DTT for 30 minutes (Make fresh for day of use)

6. Wash with dH2O 3 times, 5-10 minutes each wash

7. Incubate with Silver Nitrate (0.6%) for 30 mins and 2 mins (Make fresh for day of use)

8. Rinse with dH2O 3 times, 5-10 minutes each rinse

9. Incubate with Developer for 5 minutes

10. Incubate with fresh developer for 5-10 mins: WATCH GEL CAREFULLY

11. Add stop solution and incubate for 5-10 mins (Store at room temperature)

12. Store in preserving solution (incubate for 10 minutes)
   
Coomassie Blue Stain
   

This is a permanent, sensitive staining of proteins

NOTE: Simplyblue from Invitrogen is an excelent substitute for Coomassie staining. Simplyblue requires no toxic reagents like methanol and acetic acid, but it is faster and more sensitive than Coomassie. All that is required is washing the gel 3X for 5 minutes after running, then a 1 hour incubation with the dye followed by a couple of washes with water (1-3 hours). Invitrogen claims sensitivities as high as <3 ng when destaining overnight

1. Wash gel throughly with dH2O

2. Stain with Coomasie Blue for at least 30 minutes to 1 hr

3. Add Destain until the background is gone and blue bands are clearly visible. Changing the Destain solution can help speed up the process. Adding Kimwipes can also speed up the process

4. Store in Preserving solution