Coomassie Blue Staining
TIP: All washes and incubations are done with constant, gentle shaking.
- Immerse gel in 50% ethanol/10% acetic acid for at least 1 hr.
- Soak in 5% ethanol/5% acetic acid overnight or for a minimum of 2 hours.
- Wash in diH2O for 1 hr.
- Add Gel-Code Blue Stain reagent (Pierce, #24592) for at least 3 hrs.
- Wash in diH2O twice, 15 min each.
- Rinse in diH2O for 1 hr.
- 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.
Solution A |
Solution B |
||||||
diH2O (ml) |
10 N NaOH (ml) |
NH4OH (ml) |
Silver Nitrate (g) |
diH2O (ml) |
Final Volume (ml) | ||
21 |
0.2 |
1.3 |
1.5 |
4 |
100 | ||
31.5 |
0.3 |
1.95 |
2.25 |
6 |
150 | ||
105 |
1 |
6.5 |
7.5 |
20 |
500 | ||
210 |
2 |
13 |
15 |
40 |
1000 | ||
315 |
3 |
19.5 |
22.5 |
60 |
1500 | ||
420 |
4 |
26 |
30 |
80 |
2000 |
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
- Cut gels at the top/basic corner.
- Immerse in 50% ethanol/10% acetic acid for at least 1 hr.
- Soak in 5% ethanol/5% acetic acid overnight for a minimum of 2 hours to several days.
- Wash in diH2O for 1 hr.
- Fix in 1% glutaraldhyde/0.5 M sodium acetate for 30-45 min.
- Wash in diH2O, three times for 15 min each.
- Add Gel-Code Blue Stain reagent (Pierce, #24592) for at least 3 hrs.
- Wash in diH2O twice for 15 min each.
- Wash in diH2O for 1 hr.
- Stain gel in ammonical silver nitrate solution for 1 hr.
- Rinse gel three times for 5 min each in diH2O.
- Develop gel in developing solution for 5-10 min or until signal to noise is appropriate.
- Stop development by adding 100-150 ml of the stop solution.
- To re-stain, rinse gel in diH2O three times for 15 min each, then repeat starting from the incubation with silver nitrate.
2. Destaining
- If the gel is over-developed, it can be destained by adding the destaining solution to the gel for 30 sec-5 min.
- The silver staining should fade away slowly.
- At the right moment, scan the gel and save.
- Otherwise, rinse extensively in diH2O five or six times.
- 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.
- Rinse gels in HPLC grade H2O for 5 min.
- Equilibrate gel in 1% sodium carbonate for 15 min.
- Incubate gels in imidazole/SDS solution for 30 min.
- Rinse gels in HPLC grade H2O for 1 min.
- Develop gels in zinc acetate solution for 1-5 min or until spots are well resolved.
- Rinse gels in HPLC grade H2O three times for 5 min each.
- Spots can be cut out of the gels for sequencing, or
- 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