All solutions specified in the following should be prepared fresh.
1. Conventional Staining Methods
1.1. Silver Staining Protocol by Heukeshoven and Dernick (Modified)
The protocol uses silver nitrate for impregnation and thus belongs to the acidic methods. The sequence of the procedure is as follows (use approx 2.5 L of each solution for simultaneous staining of 10 large gels [20 × 25 cm]); all steps have to be performed with shaking:
a) Fix the gel in fixing solution for at least 2 h or overnight.
b) Sensitize for 2 h in sensitizing solution.
c) Wash the gel using Milli-Q water, 2 × 20 min.
d) Impregnate with silver staining solution for 30 min.
e) Wash the gel using Milli-Q water for a few seconds.
f) Incubate the gel prior to development in incubating solution for 1 min.
g) Develop the gel for 5–20 min in developing solution.
h) Stop the reaction using stop solution with shaking for approx 15 min.
1.2. Silver Staining Protocol by Hochstrasser
This method (http://us.expasy.org/ch2d/protocols/protocols.fm4.html) is representative of basic silver staining methods and offers extremely high sensitivity. The sequence of the procedure is as follows (use approx 2.5 L of each solution for simultaneous staining of 10 large gels [20 × 25cm]); all steps have to be performed with shaking:
a) Fix the gel in fixing solution I for 1 h.
b) Fix the gel in fixing solution II for at least 2 h or overnight.
c) Wash the gel using Milli-Q water for 5 min.
d) Sensitize for 2 h in sensitizing solution I.
e) Wash the gel using Milli-Q water, 3 × 10 min.
f )Sensitize for 2 h in sensitizing solution II, 2 × 30 min.
g) Wash the gel using Milli-Q water, 4 × 4 min.
h) Impregnate with silver staining solution for 30 min.
i) Wash the gel using Milli-Q water, 4 × 4 min.
j) Develop the gel for 5–10 min in developing solution.
k) Stop the reaction using stop solution with shaking for approx 15 min.
2. MALDI-TOF-Compatible Staining Methods
2.1. Silver-Staining Protocol by Sinha et al.
Our protocol has been designed to combine high sensitivity, minimal protein-toprotein variation, and high reproducibility, with the possibility of identifying excised protein spots using MALDI-TOF MS. A MALDI-compatible silver stain is especially recommended, if the amount of protein extract is limited. With this method, only the surface of the proteins is stained, and protein loads as small as 100 μg per gel can be used. Development can be extended to very long periods, making the procedure extremely reproducible from batch to batch. Take care that stringent conditions of cleanliness are maintained to reduce the risk of keratin contamination.
The sequence of the procedure is as follows (use approx 2.5 L of each solution for simultaneous staining of 10 large gels [20 × 25 cm]); all steps have to be performed with shaking:
a) Fix the gel in fixing solution for at least 1 h, change the solution, and fix overnight or incubate the gel in fixing solution 4 × 30 min.
b) Sensitize for 45 min in sensitizing solution.
c) Wash the gel using Milli-Q water, 6 × 10 min.
d) Impregnate with silver staining solution for 30 min.
e) Wash the gel using Milli-Q water for a maximum of 15 s.
f) Develop the gel for 30–40 min in developing solution.
g) Stop the reaction using stop solution with shaking for approx 45 min.
h) Wash the gel using Milli-Q water for 2 × 30 min.
2.2. Silver Staining Protocol by Yan et al.
This method is a modification of the commercial kit Silver Stain PlusOne (Amersham Biosciences), which is based on the original silver staining protocol by Heukeshoven and Dernick. By omitting glutaraldehyde in the sensitization step and formaldehyde in the silver impregnation step, the staining protocol is compatible with MALDI and ESI.
The sequence of the procedure is as follows (use approx 2.5 L of each solution for simultaneous staining of 10 large gels [20 × 25cm]); all steps have to be performed with shaking:
a) Fix the gel in fixing solution for 2 × 15 min.
b) Sensitize for 2 h in sensitizing solution.
c) Wash the gel using Milli-Q water, 3 × 5 min.
d) Impregnate with silver staining solution for 30 min.
e) Wash the gel using Milli-Q water for 2 × 1 min.
f) Develop the gel for 5–20 min in developing solution.
g) Stop the reaction using stop solution with shaking for approx 15 min.
h) Wash the gel using Milli-Q water for 3 × 5 min.
2.3. Silver Oxidation of Excised Protein Spots
Removal of silver ions prior to tryptic digestion is recommended for successful protein identification. The method described by Gharadaghi et al. is as follows:
a) Add a small volume of a 1:1 solution potassium ferricyanide (30 mM) and sodium thiosulphate (100 mM) to the gel piece.
b) Stir frequently until the dark color disappears.
c) Wash three to five times with Milli-Q water to stop the reaction.
d) Discard the water and cover the gel piece with 200 mM ammonium bicarbonate buffer for about 20 min.
e) Wash with water.
f) Discard water and cover the gel piece with acetonitrile.
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Reference
- Walker, J. M. (Ed.). (2005). The proteomics protocols handbook. Humana press.