Silver Staining of Gels

Procedure:

1) Load gel with 1 l per lane of 1/10 dilution of molecular weight stds, and no more than 0.I g per lane of purified sample. Run minigel at.

2) After run, immerse gel in a solution of .place in a glass or plastic container reserved for silver staining,

3) Wash plate three times (ELISA plate washer should be used) with PBS-T and empty wells. Add 100 l of primary antibody diluted in PBS-T containing 1% BSA. In controls, use PBS-T/1% BSA alone or control antibody at same dilution or g/ml concentration. Incubate overnight at 4°C.

4) Wash five times with PBS-T and empty wells. Add 100 l of secondary antibody diluted (usually 1/1000) in PBS-T containing 1% BSA or 1% normal goat serum. Incubate for 1 hr at 37°C.

5) Wash five times with PBS-T and empty wells. Add 100 l of ABTS/H2O2 (mix 1 l of H2O2 with 1 ml of ABTS immediately prior to use) and incubate 10 - 20 or up to 60 min at RT on a shaker platform. Read plate at 405 - 414 nm.

Reagents

Carbonate-Bicarbonate Coating Buffer, pH 9.6 (500 ml) Na2CO3 0.795 gm NaHCO3 1.46 gm Thimerosol 0.05 gm

PBS-T (1000 ml) NaCl 8 gm KH2PO4 0.2 gm Na2HPO4 1.15 gm KCl 0.2 gm Tween 20 1 ml Thimerosol 0.1 gm

ABTS (80 ml) citric acid monohydrate 0.49 gm Na2HPO4 0.47 gm ABTS 44 mg

Store solution in a brown bottle; discard solution when no longer clear. ABTS is: 2, 2'azino-di-(3-ethylbenzthiozoline sulfonic acid); Sigma#A-1888.

Link: http://people.virginia.edu/~gwl6s/protocols/silverstain.html

Silver Staining SDS PAGE Gels

Non-Kit based Silver staining Method:

Materials Needed:

1. Silver Nitrate
2. 1% Citric Acid: 100 ml of DI water + 1 g of Citric Acid
3. 30 % NaOH(7.5 M): 100 ml of DI water + 30 g of NaOH
4. 14.8 M Ammonium Hydroxide
5. 38% Formaldehyde
6. Ultra-pure water, use this for all steps and reagents
7. 50% Aqueous Glutaraldehyde (optional)
8. Glass tray or Novex Stain Ease Gel tray. If using glass, make sure to clean well with soap and DI water. If using Novex tray, use only trays designated for use with silver staining do not use trays which have been used for coomassie staining

Method:

1. Make 7% Acetic Acid: 186 ml of water + 14 ml of acetic acid
2. Make 50% Methanol: 200 ml of water + 200 ml of methanol. Optional for extra fixation/crosslinking add 240 l of 50% glutaraldehyde to the 50% methanol (makes solution 0.03% glutaraldehyde).
3. Soak gel in 7% acetic acid for 7 minutes.
4. Soak gel in 200 ml of 50% methanol for 20 minutes.
5. Soak gel in 200 ml of 50% methanol for 20 minutes.
6. Prepare Solution A: 0.8 g of silver nitrate + 4 ml of water
7. Rinse gel in ~200 ml water for 10 minutes
8. Rinse gel in ~200 ml water for 10 minutes

Note: Steps 7 and 8 are very important for the NuPAGE gels if you skip these steps or do not rinse the gel for long enough the gel will develop too quickly and have significantly more background.

9. 5 minutes before end of final water rinse prepare solution B: 21 ml of water + 250 ul of 30% NaOH (to make 0.36%) + 1.4 ml of 14.8M ammonium hydroxide
10. To make staining solution: add solution A to solution B dropwise while stirring then add 76 ml of water.
11. Soak gel in the staining solution for 15 minutes
12. Rinse gel in ~200 ml water for 5 minutes
13. Rinse gel in ~200 ml water for 5 minutes
14. Make developing solution: 200 ml of water + 1 ml of 1% citric acid + 100 ul of 37% formaldehyde
15. Soak gel in developing solution until bands are visible usually 2 to 15 minutes
16. Stop development by rinsing gel with 3 changes of ~200 ml water

The sensitivity of this method should be in the 10ng/band range.

Link: http://www.fhcrc.org/science/labs/hahn/methods/biochem_meth/silver_stain.html

Rapid Silver staining

1. Fixation (30% Methanol and 5% Acetic acid): Add 50ml of fixing solution for 10 min
2. Water washes: Several changes of water for at least half an hour
3. Hypo (10mg/100ml Sodium thiosulfate): Add Hypo for one min and quickly wash the gel with water
4. Silver nitrate (0.1 gm with 100 l Formaldehyde): Add 50 ml of the silver nitrate for 8-10 min
5. Developer (1.5% with 50 l Formaldehyde): Quickly wash the gel with developer to remove the excess stain and gently rinse the gel in Developer until bands appear
6. Stop solution (2M citric acid): Add 5 ml of stop solution

Link: http://www.protocol-online.org/prot/Detailed/3697.html

Better to just buy a kit. I used to make my own reagents for years, but the commercial kits are faster, more sensitive, less variable and even cheaper when you figure in time.

The kits from Pierce and Invitrogen are the best.

I use a silver staining kit from GE Healthcare for 2D electrophoresis and IEF. But according to the handbook the procedures are different for each electrophoresis. For example acetic acid is used to fix 2D gels and TCA is used to fix IEF gel. Why?

Another problem is, my IEF gel always forms this black line, on the spot where I put my acidic electrode strip, after staining. Any suggestions?

Thanks a lot.