This experiment describes the process of gel staining silver staining. This experiment is from the Protein Purification and Identification Laboratory Guide by Zhu Houzhu.

Operation method

silver dyeing experiments

Materials and Instruments

Methanol (50% and 5%v v) Dithiothreitol AgNO3 Citric Acid (Solid) Developer

Move

reagents

Methanol (50% and 5% v/v)

10umol/L dithiothreitol (DTT)

_AgNO3 (0.1%w/v)

Citric acid (solid)

Developer

Operating Procedures

This operating procedure was modified from the method of Merril (1987). The gel was shaken slowly on a rotating platform at room temperature and the following steps were performed.

1) Soak in 50% methanol solution twice for 15 min each time.

2)Soak in 5% methanol solution for 10 min.

3)Rapidly wash with _H2O for 3 times.

4)Soak in 10umol/L DTT solution for 20 min.

5)Soak in 0.1% AgNO3 solution for 20 min.

6)Wash with _H20 for 1 time quickly, and then wash with developing solution for 2 times, each time not more than 15s.

7)Soak in developer solution for several minutes until protein bands are visible.

8)Add solid citric acid (dihydrate) (about 10 g of citric acid per 200 ml of developer) to stop the development.

9)Cover the gel with aluminum foil (so as to avoid light) and continue to soak for 10 min.

10)Wash thoroughly with _H2O. The gel was soaked under aluminum foil (in the dark) in H2O for 10 min. and then placed (in the dark room) in _H20 overnight to develop the protein bands.

If required, the gel can be soaked in 4% glycerol for >lh and then dried.

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