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Gel Stain Reagents (Silver Staining)

This reagents stain proteins in gel with silver after electrophoresis (PAGE). Compared with Coomassie Brilliant Blue (CBB) staining, high sensitive staining can be done. Depending on the staining / destaining times, they detect a few nanograms level of protein.

Principles

Silver ions are considered to combine selectively to proteins at specific constituents such as sulfhydryl (-SH), amino (-NH2), hydroxyl (-OH), and carboxyl (-COOH) groups, especially at the sulfhydryl group. This is because an acid-mediated conformational change in the protein exposes the sulfhydryl binding, making the sulfhydryl group more reactive with silver ions than the other groups. Consequently, silver ions complexed with ammonium ions in the reagent combine with proteins by substituting the sulfhydryl groups of the protein with ammonium ions.

Silver Stain 2 Kit wako

Silver Stain 2 Kit wako is a general silver staining kit staining protein bands in approximately 70 minutes. The staining density can be freely adjusted with the included stopping solution.

Gel: SuperSep™ Ace 12.5%, 12-well
Sample: E. coli crude extract

Kit composition

100 mL each x 1

  • Fixative stock solution (methanol, acetic acid)
  • Enhancing stock solution (dithiothreitol, glutaraldehyde)
  • Staining solution A (silver nitrate)
  • Staining solution B (ammonia, sodium hydroxide)
  • Developer stock solution (formaldehyde, citric acid)
  • Stop solution (citric acid)

Silver Stain MS Kit

Silver Stain MS Kit is a silver staining kit optimized for mass spectrometry. Conventional silver staining kits are not suitable for mass spectrometry, because glutaraldehyde in the enhancer cross-links amino groups, thus reducing the efficiency of in-gel digestion. This product is glutaraldehyde-free and is effective for mass spectrometry. In addition to mass spectrometry, it is useful for staining after SDS-PAGE due to its higher sensitivity. A destaining solution, which is used after band excision, is also included.

Kit composition

  • Enhancing stock solution: 200 mL
  • Staining stock solution: 200 mL
  • Developing stock solution: 100 mL
  • Developing powder: 20 g
  • Stop Solution: 200 mL
  • Destaining solution A: 50 mL
  • Destaining solution B: 50 mL

Application Data

Rabbit phosphorylase was separated by SDS-PAGE and stained using the Silver Stain MS Kit. After staining, the bands were excised and digested with trypsin in gel for MALDI-TOF/MS.

Data Source:Dr. Yoshinao Wada, Osaka Women's and Children's Hospital

Table for kit comparison

Operating procedure Composition Silver Staining Kit
MS
Glutaraldehyde Contained Not contained
Gel after SDS-PAGE
1.Fixation-1 Fixative solution-1*1 10 min 20 min
2.Fixation-2 Fixative solution-2*1 10 min 10 min
3.Washing with water (Deionized water) 10 min
4.Enhancement Enhancing solution 10 min 1 min
5.Washing with water (Deionized water) 5 min 1 min × 2
6.Silver staining Staining solution 15 min 20 min
7.Washing with water (Deionized water) 2 to 5 min × 3 1 min x 2
8.Development Developing solution 5 min 3~10 min
9.Stopping development Stop solution 2~3 min 1 min
10.Washing with water (Ionized water) 2 min x 3 1 min x 3
Total time Approximately 70min Approximately 70 min
Gel excision*2
11.Destaining 15 min
In-gel digestion*3
Mass spectrometry
  • *1 As this fixative solution is not included among the constituent reagents, you will need to prepare it yourself.
  • *2 A brown fraction is excised.
  • *3 The following reagents are commercially available as in-gel digestion enzymes for proteome research:
     Product Number: 125-05061 Lysyl endopeptidase, mass spectrometry grade 20 μg × 5
     Product Number: 202-15951 Trypsin, porcine pancreas-derived, mass spectrometry grade 20 μg × 5

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Enzymes for in-gel Digestion in Proteome Research

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