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Features

High sensitivity – 0.05 ng of protein per band can be detected (Fig.2) when the conventional protocol is used.
Clear background (Fig.1).
Fast and easy staining – procedure takes only 60 minutes when the fast protocol is used.
Compatible with in situ zymography.

Applications

Highly sensitive staining of proteins, DNA and RNA in polyacrylamide gels.
Stains both non-denaturing or denaturing polyacrylamide gels containing SDS or urea.
Visualization of nanogram quantities of DNA and RNA.

Description

The PageSilver Silver Staining Kit is a complete system for extremely sensitive and rapid staining of protein, DNA, RNA in polyacrylamide gels. The procedure is based on the use of silver nitrate to bind proteins at a weakly acidic pH and subsequent reduction of silver ions to metallic silver by formaldehyde at an alkaline pH (1, 2, 3). The staining procedure is fast, simple and can be completed in approximately 60 minutes with very little hands-on time. All components of the PageSilver Silver Staining Kit are optimized for maximum sensitivity of protein staining (Fig.2) with a clear background (Fig.1). As little as 0.05-0.6 ng of protein per band can be visualized. This reflects a >100 times higher sensitivity compared to traditional Coomassie Brilliant Blue R250 staining and allows for superior detection of low abundance proteins.

Quality Control

The kit is tested using the staining protocol for maximum speed, different dilutions of BSA (0.1-2.0 ng) are electrophoresed on a 1.0 mm. 12% Tris-glycine SDS gel. Silver staining detects 0.1 ng BSA in clear background.

Components

Sensitizer concentrate
Staining reagent
Developing reagent
Formaldehyde
Stop reagent
Detailed Protocol

Note

Analysis of stained proteins by mass spectrometry is not recommended.

Highly sensitive protein staining with PageSilver™ Silver Staining Kit

Figure 1. Highly sensitive protein staining in a clear background with PageSilver™ Silver Staining Kit.
Decreasing amounts of cell lysates were separated on 4-12% Tris-glycine SDS-PAGE and stained with the PageSilver™ Silver Staining Kit.
M – PageRuler™ Plus Prestained Protein Ladder
1, 4 – 500 ng of total protein
2, 5 – 200 ng of total protein
3, 6 – 100 ng of total protein

Figure 2. High sensitivity of staining with PageSilver™ Silver Staining Kit.
Different amounts of BSA were run on 4-12% Tris-glycine SDS-PAGE and stained with the PageSilver™ Silver Staining Kit according to protocol for maximum sensitivity.

Patents, Licenses, Trademarks

RECOMMENDATIONS FOR USE

ADDITIONAL PROTOCOLS

Gel Staining Procedure with PageSilver™ Silver Staining Kit

With microwaving (fast protocol)	Without microwaving (conventional protocol)
Total time Sensitivity
1 hour 0.1 ng/band	2 hours 40 min 0.05 ng/band
1. Gel fixing 1
Rinse the gel with deionized water Add gel fixing solution 1 Microwave for 30 s. Do not boil Fix with gentle agitation for 10 min Discard the solution	Rinse the gel with deionized water Add gel fixing solution 1 and gently agitate for 60 min Discard the solution
2. Gel fixing 2 and Washing
Perform Gel fixing 2 procedure twice: Add gel fixing solution 2 Microwave for 30 s. Do not boil Fix with gentle agitation for 10 min Discard the solution Perform Washing procedure twice: Add deionized water and gently agitate for 20 s Discard the water	Perform Gel fixing 2 procedure three times: Add gel fixing solution 2 and gently agitate for 20 min Discard the solution Perform Washing procedure twice: Add deionized water and gently agitate for 20 s Discard the water
3. Sensitizing and Washing
Add sensitizing solution and gently agitate for 1 min Discard the solution Perform Washing procedure twice: Add deionized water and gently agitate for 20 s Discard the water	Add sensitizing solution and gently agitate for 1 min Discard the solution Perform Washing procedure twice: Add deionized water and gently agitate for 20 s Discard the water
4. Staining and Washing
Add staining solution and gently agitate for 20 min Discard the solution Perform Washing procedure twice: Add water and gently agitate for 20 s Discard the water	Add staining solution and gently agitate for 20 min Discard the solution Perform Washing procedure twice: Add water and gently agitate for 20 s Discard the water
5. Developing
Add developing solution and gently agitate for ~4 min Discard the solution	Add developing solution and gently agitate for 5-10 min Discard the solution
6. Terminating
Add stop solution and gently agitate for 5 min Discard the solution	Add stop solution and gently agitate for 10 min Discard the solution

Protein Samples: Preparation for Loading on SDS-PAGE

Step	Sample preparation with the DualColor™ Protein Loading Buffer Pack	Sample preparation with the Protein Loading Buffer Pack
Thaw	Thaw the buffer pack components either at room temperature or at 37°C for a few minutes to dissolve precipitates.
Mix	Vortex gently, but thoroughly to ensure that the solution is homogeneous.
Dilution	2.0 µl of 20X Reducing Agent	2.5 µl of 20X Reducing Agent
	Protein sample (~0.5 ng – 2.5 µg) For Western blots or gels to be treated with Coomassie based stains, use up to 2.5 µg of total protein per minigel well. For silver staining applications use up to 10 ng of total protein per minigel well.
	10 µl of 4X DualColor™ Protein Loading Buffer Water, nuclease-free to 40 µl*	10 µl of 5X Protein Loading Buffer Water, nuclease-free to 50 µl*
Denaturation	Incubate samples at 95-100°C for 5 minutes.
Loading	Spin for a few seconds in a microcentrifuge. Apply directly to an SDS-polyacrylamide gel. Use ~10 µl per minigel well.

* The sample volume can be scaled up or down.

Staining of Small Proteins

Protein fixation with glutaraldehyde is required before staining the gel with Coomassie Brilliant Blue dye (PageBlue™ Protein Staining Solution, #R0571) or silver (PageSilver™ Silver Staining Kit, #K0681).

Note

Other compounds commonly used for protein fixation (e.g., acetic acid, isopropanol, ethanol, TCA) are not suitable; proteins will wash away during staining procedure.

Procedure

Add 100 ml of deionized or distilled water to the gel and wash for 1 min with gentle agitation. Discard the wash.
Add 50 ml of freshly prepared 5% glutaraldehyde solution (gel should be covered completely). Fix with gentle agitation for 30 min. Discard the solution.
Add 100 ml of deionized or distilled water to the gel and wash for 5 minutes with gentle agitation. Discard the wash. Repeat this step twice. The gel is now ready for staining.

For detailed staining protocols using Fermentas PageBlue™ Protein Staining Solution or PageSilver™ Silver Staining Kit, refer to the product manuals.

Note

Proceed directly to the staining step in PageBlue™ Protein Staining Solution protocol and to sensitizing step in PageSilver ™ Silver Staining Kit protocol.

Rabilloud, T. et al., Silver-staining of proteins in polyacrylamide gels: a general overview, Cell. Mol. Biol., 40, 57-75, 1994. Cell. Mol. Biol. 40, 57-75, 1994.
Blum, H., Beier, H. And Gross, H.J., Improved silver staining of plant proteins, RNA and DNA in polyacrylamide gels, Electrophoresis, 8, 93-99, 1987.
Sorense, B.K., et al., Silver Staining of Proteins on Electro-blotting Membranes and Intensification of Silver Staining of Proteins Separated by Polyacrylamide Gel Electrophoresis, Anal.Biochem., 304, 33-41, 2002.