Protein Staining Procedures

Coomassie Blue Staining

TIP: All washes and incubations are done with constant, gentle shaking.

1. Immerse gel in 50% ethanol/10% acetic acid for at least 1 hr.
2. Soak in 5% ethanol/5% acetic acid overnight or for a minimum of 2 hours.
3. Wash in diH₂O for 1 hr.
4. Add Gel-Code Blue Stain reagent (Pierce, #24592) for at least 3 hrs.
5. Wash in diH₂O twice, 15 min each.
6. Rinse in diH₂O for 1 hr.
7. Gels can be stored at 4°C.

Silver Nitrate Staining

A: Solutions:

1. Ammonical silver nitrate staining solution:

Prepare solutions A and B as described below and, after mixing together, make to the final volume. Dissolve each solution WELL by vortexing vigorously.

Add solution B to solution A slowly. A brown precipitate will appear then disappear. If disappearance of the brown precipitate is slow, add drops of NH4OH. Bring to final volume.

TIP: diH₂O should be high quality. Otherwise, silver nitrate will precipitate.

2. Developing solution ( 0.1% formaldehyde, 0.01% citric acid):

100 µl formaldehyde and 0.01 g of citric acid /100 ml diH₂O

3. Stop solution (2% acetic acid):

2 ml acetic acid plus 98 ml diH₂O

4. Destaining solution:

Prepare separately:

10 mM sodium thiosulfate pentahydrate ( 0.248 g/100 ml diH₂O)
30 mM potassium ferricyanide (0.988 g/100 ml diH₂O)

Mix both solutions together.

B: Procedure:

All washes and incubations are done with constant, gentle shaking.

1. Staining

1. Cut gels at the top/basic corner.
2. Immerse in 50% ethanol/10% acetic acid for at least 1 hr.
3. Soak in 5% ethanol/5% acetic acid overnight for a minimum of 2 hours to several days.
4. Wash in diH₂O for 1 hr.
5. Fix in 1% glutaraldhyde/0.5 M sodium acetate for 30-45 min.
6. Wash in diH₂O, three times for 15 min each.
7. Add Gel-Code Blue Stain reagent (Pierce, #24592) for at least 3 hrs.
8. Wash in diH₂O twice for 15 min each.
9. Wash in diH₂O for 1 hr.
10. Stain gel in ammonical silver nitrate solution for 1 hr.
11. Rinse gel three times for 5 min each in diH₂O.
12. Develop gel in developing solution for 5-10 min or until signal to noise is appropriate.
13. Stop development by adding 100-150 ml of the stop solution.
14. To re-stain, rinse gel in diH₂O three times for 15 min each, then repeat starting from the incubation with silver nitrate.

2. Destaining

1. If the gel is over-developed, it can be destained by adding the destaining solution to the gel for 30 sec-5 min.
2. The silver staining should fade away slowly.
3. At the right moment, scan the gel and save.
4. Otherwise, rinse extensively in diH₂O five or six times.
5. Repeat silver staining.

   Note: Staining intensity may be less than normal.

Zinc Imidazole Negative Staining

A: Solutions:

1. 1% sodium carbonate:

1 g sodium carbonate/100 ml diH₂O

2. 200 mM imidazole/0.1% SDS:

1.36 g imidazole and 0.1 g SDS per 100 ml diH₂O

3. 100 mM zinc acetate:

2.2 g zinc acetate/100 ml diH₂O, filter through a 0.45 µm filter.

B: Procedure:

All washes and incubations are done with constant, gentle shaking.

1. Rinse gels in HPLC grade H₂O for 5 min.
2. Equilibrate gel in 1% sodium carbonate for 15 min.
3. Incubate gels in imidazole/SDS solution for 30 min.
4. Rinse gels in HPLC grade H₂O for 1 min.
5. Develop gels in zinc acetate solution for 1-5 min or until spots are well resolved.
6. Rinse gels in HPLC grade H₂O three times for 5 min each.
7. Spots can be cut out of the gels for sequencing, or
8. Gels can be:

• stored at 4°C up to one week without significant protein diffusion
• destained with 50 mM EDTA and washed for:
  • re-staining
  • protein transfer
  • silver staining