GE Healthcare

Instructions 71-7177-00 AL

PlusOne Silver Staining Kit, Protein

Intended use
The PlusOne Silver Staining Kit, Protein is intended for research use only, and shall not be
used in any clinical or in vitro procedures for diagnostic purposes.

Table of contents
1 Introduction .........................................................................................................................................................3
2 Kit contents and characteristics ................................................................................................................4
2.1 Kit contents........................................................................................................................................................................ 4
2.2 Technical data.................................................................................................................................................................. 4
3 Protocol and procedure for silver staining............................................................................................5
3.1
General information ...................................................................................................................................................... 5
3.2
Protocol selection ........................................................................................................................................................... 5
A ExcelGel SDS, ExcelGel XL SDS 12-14 and mini slab gels 0.5 mm ........................................................... 6
B CleanGel IEF and Immobiline DryPlate................................................................................................................. 8
C Ettan DALT 12.5 Precast gel and conventional slab gels 1.5 mm.......................................................... 10
D Conventional slab gels 1 mm................................................................................................................................. 12
E Ettan DALT 12.5 Precast gel / In-house cast 1 mm gel on glass backing (Bind-Silane treated),
MS compatible................................................................................................................................................................ 14
F Protocol for PhastGel SDS media ......................................................................................................................... 16
G Protocol for PhastGel Native media.................................................................................................................... 18
H Protocol for PhastGel IEF media ........................................................................................................................... 20
4 Troubleshooting guide................................................................................................................................. 22
5 Ordering information.................................................................................................................................... 23
5.1 PlusOne Silver Staining Kit, Protein ..................................................................................................................... 23
5.2 Chemicals and accessories..................................................................................................................................... 23
5.3 Precast gels and related products....................................................................................................................... 24
5.4 Electrophoresis equipment ..................................................................................................................................... 26

2 Instructions 71-7177-00 AL
1 Introduction
PlusOne Silver Staining Kit, Protein is a reagent kit for the fast, easy, reproducible and
sensitive staining of proteins in polyacrylamide (PAA) gels. Ready-to-use solutions and pre-
weighed chemicals minimize preparation time and maximize ease of use and reliability.
The kit, which is part of the PlusOne range of electrophoresis chemicals, is based on the
methodology of Heukeshoven and Dernick1.
The high sensitivity of the visualization technique allows detection of most proteins down
to the nanogram range, which is 100 times more sensitive than Coomassie Brilliant Blue.
The method is reliable and reproducible and gives essentially colorless backgrounds in
most gel electrophoresis systems.
PlusOne Silver Staining Kit, Protein is developed for use with GE Healthcare precast gels as
well as with thicker, conventional polyacrylamide gels. The kit is thus suitable for non-
denaturing gels, denaturing gels containing sodium dodecyl sulphate (SDS) and/or urea,
and isoelectric focusing (IEF) gels.

Fig 1. PlusOne Silver Staining Kit, Protein.

1. Simplified method for silver staining of proteins in polyacrylamide gels and the mechanism of silver staining. Electrophoresis 6 (1985)
103-112, Heukeshoven, J. and Dernick, R.

Instructions 71-7177-00 AL 3
2 Kit contents and characteristics
2.1 Kit contents
PlusOne Silver Staining Kit, Protein contains the following items.

Item Quantity
Sodium acetate 10 x 17 g packets
5% Sodium thiosulphate 1 x 100 ml bottle
EDTA-Na2 10 x 3.65 g packets
Sodium carbonate 10 x 6.25 g packets
2.5% Silver nitrate 2 x 125 ml bottles
37% Formaldehyde 1 x 2.5 ml bottle
25% Glutardialdehyde 1 x 12.5 ml bottle

Quantities are sufficient to stain 10 to 60 gels depending on size and protocol used:
• 10 DALT precast gels
• 10 Precast 12.5 x 26 cm
• 20 Mini slab gels (8 to 10 x 8 cm)
• 30 to 60 PhastGel™

2.2 Technical data

Sensitivity: 0.2 to 0.6 ng protein per band
Storage: +10 to +30°C
Shelf life: 1 year at recommended storage conditions

4 Instructions 71-7177-00 AL
3 Protocol and procedure for silver staining
3.1 General information
Wear gloves at all times. Perform all steps at 21 to 25°C with constant gentle agitation on
a shaker.
The quality of the water used for making up the reagents and for washing the gel will affect
the staining result. For best results use water with a conductivity of 5 MΩ or more (e.g. Milli-
Q™ water).
Freshly made solutions (not older than 24 hours) give best results.

Precautions: The chemicals in this kit should not be discarded via public waste water
systems. Please dispose of these chemicals properly. Consult your local regulations for
more information.
Read the warning text on the label of each bottle and packet.

3.2 Protocol selection

Protocols are optimized for precast gels from GE Healthcare as well as for conventional slab
gels (Laemmli).

Type of gel Protocol Page

ExcelGel™ SDS, ExcelGel XL SDS 12-14, and mini slab gels 0.5 mm A 6
CleanGel™ IEF and Immobiline™ DryPlate B 8
Ettan™ DALT 12.5 Precast gel and conventional slab gels 1.5 mm C 10
Conventional slab gels 1 mm D 12
Ettan DALT 12.5 Precast gel / In-house cast gel on backing 1 mm, E 14
MS compatible
PhastGel SDS F 16
PhastGel Native G 18
PhastGel IEF H 20

Instructions 71-7177-00 AL 5
A ExcelGel SDS, ExcelGel XL SDS 12-14 and mini slab gels 0.5 mm
Chemicals required
• Chemicals included in this PlusOne Silver Staining Kit, Protein
• Ethanol
• Glacial acetic acid
• Glycerol (87% w/w) (1000 ml) (Code No. 17-1325-01)

Reagents
Make up the following reagent solutions using the chemicals listed above.
250 ml solution is needed per gel and step except for ExcelGel XL SDS 12-14, which requires
400 ml per gel and step and for Mini slab gels which require 125 ml solution per gel and
step.
Note: Glutardialdehyde and formaldehyde should be added immediately before use.

Reagent name Component and quantity

Fixing solution 40% Ethanol Ethanol 100 ml
Glacial acetic acid 25 ml
Water to 250 ml
Sensitizing solution Ethanol 75 ml
Sodium thiosulphate (5% w/v) 10 ml
Sodium acetate (17 g) 1 packet
Water to 250 ml
Before use: Add 1.25 ml glutardialdehyde (25% w/v)
Silver solution Silver nitrate solution (2.5% w/v) 25 ml
Water to 250 ml
Developing solution Sodium carbonate (6.25 g) 1 packet
Water to 250 ml
Stir vigorously to dissolve the sodium carbonate
Before use: Add 0.2 ml formaldehyde (37% w/v)
Stop solution EDTA-Na2•2H2O (3.65 g) 1 packet
Water to 250 ml
Washing solution Water
Preserving solution for plastic backed Glycerol (87% w/w) 25 ml
gels Water to 250 ml
Preserving solution for gels not Ethanol 75 ml
supported on plastic films Glycerol (87% w/w) 11.5 ml
Water to 250 ml

6 Instructions 71-7177-00 AL
 Staining procedure
All steps should be performed with gentle shaking of the staining tray.

1 Fixation: 30 min.
Soak the gel in fixing solution for 30 minutes.
2 Sensitizing: 30 min.
Remove the solution. Add sensitizing solution and leave shaking for at least 30 minutes.
3 Washing: 3 x 5 min.
Remove the sensitizing solution. Add distilled water and wash three times for 5 minutes
each time.
4 Silver reaction: 20 min.
Add silver solution and leave shaking for 20 minutes.
5 Washing: 2 x 1 min.
Remove the silver solution. Rinse twice in distilled water for one minute each time.
6 Developing: 2 to 5 min.
Add developing solution and leave shaking for 2 to 5 minutes. Transfer the gel to
stopping solution when the bands / spots have reached desired intensity.
7 Stopping: 10 min.
Leave gel shaking in stopping solution for 10 minutes.
8 Washing: 3 x 5 min.
Remove the stop solution. Add distilled water and wash three times for 5 minutes each
time.
9 Preserving:
20 min. (plastic-backed gels)
2 x 30 min. (gels not supported on plastic films)
Add preserving solution and leave shaking for 20 minutes for plastic backed gels. For
gels not supported on plastic films, shake for 30 minutes, pour off the solution, add
fresh and shake for a further 30 minutes.
10 Drying: Overnight
Put the gel on a glass plate and wrap it in Cellophane sheet. Leave the gel to dry
overnight at room temperature. Do not put the gel in a heating cabinet (the silver stain
bleaches at elevated temperatures).

Instructions 71-7177-00 AL 7
B CleanGel IEF and Immobiline DryPlate
Chemicals required
• Chemicals included in this PlusOne Silver Staining Kit, Protein
• Ethanol
• Trichloroacetic acid (TCA)
• Glycerol (87% w/w) (1000 ml) (Code No. 17-1325-01)

Reagents
Make up the following reagent solutions using the chemicals listed above.
250 ml solution is needed per gel and step.
Note: Glutardialdehyde and formaldehyde should be added immediately before use.

Reagent name Component and quantity

Fixing solution TCA Trichloroacetic acid 50 g
Water to 250 ml
Sensitizing solution Ethanol 75 ml
Sodium thiosulphate (5% w/v) 10 ml
Sodium acetate (17 g) 1 packet
Water to 250 ml
Before use: Add 1.25 ml glutardialdehyde (25% w/v)
Silver solution Silver nitrate solution (2.5% w/v) 25 ml
Water to 250 ml
Developing solution Sodium carbonate (6.25 g) 1 packet
Water to 250 ml
Stir vigorously to dissolve the sodium carbonate
Before use: Add 0.2 ml formaldehyde (37% w/v)
Stop solution EDTA-Na2•2H2O (3.65 g) 1 packet
Water to 250 ml
Washing solution Water
Preserving solution Glycerol (87% w/w) 25 ml
Water to 250 ml

8 Instructions 71-7177-00 AL
 Staining procedure
1 Fixation: 30 min.
Soak the gel in fixing solution for 30 minutes.
2 Washing: 3 x 5 min.
Wash the gel three times in distilled water for 5 minutes each time.
3 Sensitizing: 30 min.
Remove the solution. Add sensitizing solution and leave shaking for at least 30 minutes.
4 Washing: 3 x 10 min.
Remove the sensitizing solution. Add distilled water and wash 3 times for 10 minutes
each time.
5 Silver reaction: 20 min.
Add silver solution and leave shaking for 20 minutes.
6 Washing: 2 x 1 min.
Remove the silver solution. Rinse twice in distilled water for one minute each time.
7 Developing: 2 to 5 min.
Add developing solution and leave shaking for 2 to 5 minutes. Transfer the gel to
stopping solution when the bands / spots have reached desired intensity.
8 Stopping: 10 min.
Leave gels shaking in stopping solution for 10 minutes.
9 Washing: 3 x 5 min.
Remove the stop solution. Add distilled water and wash three times for 5 minutes each
time.
10 Preserving: 20 min.
Add preserving solution and leave shaking for 20 minutes.
11 Drying: Overnight
Put the gel on a glass plate and wrap it in Cellophane Sheet. Leave the gel to dry
overnight at room temperature. Do not put the gel in a heating cabinet (the silver stain
bleaches at elevated temperatures).

Instructions 71-7177-00 AL 9
C Ettan DALT 12.5 Precast gel and conventional slab gels 1.5 mm
For staining large scale gels we recommend using the Staining Tray Set (see Section
5.2 Chemicals and accessories).

Chemicals required
• Chemicals included in this PlusOne Silver Staining Kit, Protein
• Ethanol
• Glacial acetic acid
• Glycerol

Reagents
Make up the following reagent solutions using the chemicals listed above.
250 ml solution is needed per gel and step.
Note: Glutardialdehyde and formaldehyde should be added immediately before use.

Reagent name Component and quantity

Fixing solution 30% Ethanol Ethanol 75 ml
Glacial acetic acid 25 ml
Water to 250 ml
Sensitizing solution Ethanol 75 ml
Sodium thiosulphate (5% w/v) 10 ml
Sodium acetate (17 g) 1 packet
Water to 250 ml
Before use: Add 1.25 ml glutardialdehyde (25% w/v)
Silver solution Silver nitrate solution (2.5% w/v) 25 ml
Water to 250 ml
Developing solution Sodium carbonate (6.25 g) 1 packet
Water to 250 ml
Stir vigorously to dissolve the sodium carbonate
Before use: Add 0.2 ml formaldehyde (37% w/v)
Stop solution EDTA-Na2•2H2O (3.65 g) 1 packet
Water to 250 ml
Washing solution Water
Preserving solution Glycerol (87% w/w) 25 ml
Water to 250 ml

10 Instructions 71-7177-00 AL
 Staining procedure
All steps should be performed with gentle shaking of the staining tray.

1 Fixation: 120 min.

Soak the gel in fixing solution for 120 minutes (or over night).
2 Sensitizing: 120 min.
Remove the solution. Add sensitizing solution and leave shaking for 120 minutes.
3 Washing: 5 x 15 min.
Remove the sensitizing solution. Add distilled water and wash five times for 15 minutes
each time.
4 Silver reaction: 120 min.
Add silver solution and leave shaking for 120 minutes.
5 Washing: 2 x 1 min.
Remove the silver solution. Wash twice in distilled water for one minute each time.
6 Developing: 4 to 6 min.
Add developing solution and leave shaking for 4 to 6 minutes. Transfer the gel to
stopping solution when the bands / spots have reached desired intensity.
7 Stopping: 120 min.
Leave shaking for 120 minutes.
8 Preserving: 120 min.
Add preserving solution and leave shaking for 120 minutes.
9 Drying: Overnight
Use the Easy Breeze™ air gel drying system gel frames to put the gel between wet
cellophane sheets (see Section 5.2 Chemicals and accessories). Leave the gel to dry
overnight at room temperature. Do not put the gel in a heating cabinet (the silver stain
bleaches at elevated temperatures).

Instructions 71-7177-00 AL 11
D Conventional slab gels 1 mm
For staining large scale gels we recommend using the Staining Tray Set (see Section
5.2 Chemicals and accessories).

Chemicals required
• Chemicals included in this PlusOne Silver Staining Kit, Protein
• Ethanol
• Glacial acetic acid
• Glycerol (87% w/w) (1000 ml) (Code No. 17-1325-01)

Reagents
Make up the following reagent solutions using the chemicals listed above.
250 ml solution is needed per gel and step.
Note: Glutardialdehyde and formaldehyde should be added immediately before use.

Reagent name Component and quantity

Fixing solution 30% Ethanol Ethanol 75 ml
Glacial acetic acid 25 ml
Water to 250 ml
Sensitizing solution Ethanol 75 ml
Sodium thiosulphate (5% w/v) 10 ml
Sodium acetate (17 g) 1 packet
Water to 250 ml
Before use: Add 1.25 ml glutardialdehyde (25% w/v)
Silver solution Silver nitrate solution (2.5% w/v) 25 ml
Water to 250 ml
Developing solution Sodium carbonate (6.25 g) 1 packet
Water to 250 ml
Stir vigorously to dissolve the sodium carbonate
Before use: Add 0.2 ml formaldehyde (37% w/v)
Stop solution EDTA-Na2•2H2O (3.65 g) 1 packet
Water to 250 ml
Washing solution Water
Preserving solution Ethanol 75 ml
Glycerol (87% w/w) 11.5 ml
Water to 250 ml

12 Instructions 71-7177-00 AL
 Staining procedure
All steps should be performed with gentle shaking of the staining tray.

1 Fixation: 60 min.
Soak the gel in fixing solution for 60 minutes (or up to 24 hours).
2 Sensitizing: 60 min.
Remove the solution. Add sensitizing solution and leave shaking for 60 minutes (or up
to 24 hours).
3 Washing: 4 x 15 min.
Remove the sensitizing solution. Add distilled water and wash four times for 15 minutes
each time.
4 Silver reaction: 60 min.
Add silver solution and leave shaking for 60 minutes.
5 Washing: 2 x 1 min.
Remove the silver solution. Wash twice in distilled water for one minute each time.
6 Developing: 4 to 6 min.
Add developing solution and leave shaking for 4 to 6 minutes. Transfer the gel to
stopping solution when the bands / spots have reached desired intensity.
7 Stopping: 60 min.
Leave shaking for 60 minutes.
8 Preserving: 60 min.
Add preserving solution and leave shaking for 60 minutes.
9 Drying: Overnight
Use the Easy Breeze air gel drying system gel frames to put the gel between wet
cellophane sheets (see Section 5.2 Chemicals and accessories). Leave the gel to dry
overnight at room temperature. Do not put the gel in a heating cabinet (the silver stain
bleaches at elevated temperatures).

Instructions 71-7177-00 AL 13
E Ettan DALT 12.5 Precast gel / In-house cast 1 mm gel on glass
backing (Bind-Silane treated), MS compatible
For staining large scale gels we recommend using the Staining Tray Set (see Section
5.2 Chemicals and accessories).

Chemicals required
• Chemicals included in this PlusOne Silver Staining Kit, Protein
• Ethanol
• Glacial acetic acid

Reagents
Make up the following reagent solutions using the chemicals listed above.
500 ml solution is needed per gel and step.
Note: Formaldehyde should be added to the developing solution immediately before use.
By omitting the glutardialdehyde from the sensitizer and formaldehyde from the
silver solution the method becomes compatible with mass spectrometry analysis,
however at the expense of sensitivity (10 ng).

Reagent name Component and quantity

Fixing solution 30% Ethanol Ethanol 150 ml
Glacial acetic acid 50 ml
Water to 500 ml
Sensitizing solution Ethanol 75 ml
Sodium thiosulphate (5% w/v) 10 ml
Sodium acetate (17 g) 1 packet
Water to 250 ml
Silver solution Silver nitrate solution (2.5% w/v) 25 ml
Water to 250 ml
Developing solution Sodium carbonate (6.25 g) 1 packet
Water to 250 ml
Stir vigorously to dissolve the sodium carbonate
Before use: Add 0.2 ml formaldehyde (37% w/v)
Stop solution EDTA-Na2•2H2O (3.65 g) 1 packet
Water to 250 ml
Washing solution Water

14 Instructions 71-7177-00 AL
 Staining procedure
All steps should be performed with gentle shaking of the staining tray.

1 Fixation: 2 x 60 min.
Soak the gel in fixing solution for 60 minutes. Repeat step once.
2 Sensitizing: 120 min.
Remove the solution. Add sensitizing solution and leave shaking for 120 minutes.
3 Washing: 5 x 8 min.
Remove the sensitizing solution. Add distilled water and wash five times for 8 minutes
each time.
4 Silver reaction: 60 min.
Add silver solution and leave shaking for 60 minutes.
5 Washing: 4 x 1 min.
Remove the silver solution. Wash four times in distilled water for one minute each time.
6 Developing: 2 to 5 min.
Add developing solution and leave shaking for 2 to 5 minutes. Transfer the gel to
stopping solution when the bands / spots have reached desired intensity.
7 Stopping: 45 min.
Leave shaking for 45 minutes.
8 Washing: 2 x 30 min.
Remove the stop solution. Add distilled water and wash two times for 30 minutes each
time.
9 Storing: Water
Use water for up to one week and 5% acetic acid for longer periods.

Instructions 71-7177-00 AL 15
F Protocol for PhastGel SDS media
Chemicals required
• Chemicals included in this PlusOne Silver Staining Kit, Protein
• Ethanol
• Glacial acetic acid
• Glycerol (87% w/w) (1000 ml) (Code No. 17-1325-01)

Reagents
Make up the following reagent solutions using the chemicals listed above.
80 ml solution is needed per 1 to 2 gels and step.
Note: Glutardialdehyde and formaldehyde should be added immediately before use
.

Reagent name Component and quantity

Fixing solution 30% Ethanol Ethanol 75 ml
Glacial acetic acid 25 ml
Water to 250 ml
Sensitizing solution Ethanol 75 ml
Sodium thiosulphate (5% w/v) 10 ml
Sodium acetate (17 g) 1 packet
Water to 250 ml
Before use: Add 0.4 ml glutardialdehyde (25% w/v) to 80
ml of the sensitizing solution
Silver solution Silver nitrate solution (2.5% w/v) 25 ml
Water to 250 ml
Developing solution Sodium carbonate (6.25 g) 1 packet
Water to 250 ml
Stir vigorously to dissolve the sodium carbonate
Before use: Add 0.05 ml formaldehyde (37% w/v) to 80 ml
developing solution
Stop solution EDTA-Na2•2H2O (3.65 g) 1 packet
Water to 250 ml
Washing solution Water
Preserving solution Glycerol (87% w/w) 25 ml
Water to 250 ml

16 Instructions 71-7177-00 AL
 Staining procedure
Steps to be programmed into the development method file:
.

Step Time Temp

Solution IN OUT (min.) (°C)
1 Fixing solution 30% Ethanol 2 0 10 50
2 Sensitizing solution 3 91 10 50
3 Water 4 0 4 50
4 Water 4 0 4 50
5 Water 4 0 4 50
6 Water 4 0 4 40
7 Silver solution 5 91 10 40
8 Water 4 0 1 20
9 Water 4 0 1 20
10 Developing solution 6 91 1 to 52 20
11 Stop solution 7 0 10 50
12 Preserving solution 8 0 10 50
1
Please consult local regulations for information on proper disposal
2
Guideline only, need to be optimized

Instructions 71-7177-00 AL 17
G Protocol for PhastGel Native media
• Chemicals required
• Chemicals included in this PlusOne Silver Staining Kit, Protein
• Ethanol
• Trichloroacetic acid (TCA)
• Glacial acetic acid
• Glycerol (87% w/w) (1000 ml) (Code No. 17-1325-01)

Reagents
Make up the following reagent solutions using the chemicals listed above.
80 ml solution is needed per 1 to 2 gels and step.
Note: Glutardialdehyde and formaldehyde should be added immediately before use
.

Reagent name Component and quantity

Fixing solution TCA Trichloroacetic acid 50 g
Water to 250 ml
Fixing solution 30% Ethanol Ethanol 75 ml
Glacial acetic acid 10 ml
Water to 250 ml
Sensitizing solution Ethanol 75 ml
Sodium thiosulphate (5% w/v) 10 ml
Sodium acetate (17 g) 1 packet
Water to 250 ml
Before use: Add 0.4 ml glutardialdehyde (25% w/v) to 80
ml of the sensitizing solution
Silver solution Silver nitrate solution (2.5% w/v) 50 ml
Water to 250 ml
Developing solution Sodium carbonate (6.25 g) 1 packet
Water to 250 ml
Stir vigorously to dissolve the sodium carbonate
Before use: Add 0.05 ml formaldehyde (37% w/v) to 80 ml
of the developing solution
Stop solution EDTA-Na2•2H2O (3.65 g) 1 packet
Water to 250 ml
Washing solution Water
Preserving solution Glycerol (87% w/w) 25 ml
Water to 250 ml

18 Instructions 71-7177-00 AL
 Staining procedure
Steps to be programmed into the development method file:
.

Step Time Temp

Solution IN OUT (min.) (°C)
1 Fixing solution TCA 1 91 10 20
2 Fixing solution 30% Ethanol 2 0 5 50
3 Sensitizing solution 3 91 10 50
4 Water 4 0 4 50
5 Water 4 0 4 50
6 Water 4 0 4 50
7 Water 4 0 4 50
8 Water 4 0 4 40
9 Silver solution 5 91 10 40
10 Water 4 0 1 20
11 Water 4 0 1 20
12 Developing solution 6 91 1 to 52 20
13 Stop solution 7 0 10 50
14 Preserving solution 8 0 10 50
1 Please consult local regulations for information on proper disposal
2 Guideline only, need to be optimized

Instructions 71-7177-00 AL 19
H Protocol for PhastGel IEF media
Chemicals required
• Chemicals included in this PlusOne Silver Staining Kit, Protein
• Ethanol
• Trichloroacetic acid (TCA)
• Glacial acetic acid (HAc)
• Glycerol (87% w/w) (1000 ml) (Code No. 17-1325-01)

Reagents
Make up the following reagent solutions using the chemicals listed above.
80 ml solution is needed per 1 to 2 gels and step.
Note: Glutardialdehyde and formaldehyde should be added immediately before use.
Preserving solution is not necessary for Phast IEF media
.

Reagent name Component and quantity

Fixing solution TCA Trichloroacetic acid 50 g
Water to 250 ml
Fixing solution 10% Ethanol Ethanol 50 ml
Glacial acetic acid 25 ml
Water to 500 ml
Sensitizing solution Ethanol 75 ml
Sodium thiosulphate (5% w/v) 10 ml
Sodium acetate (17 g) 1 packet
Water to 250 ml
Before use: Add 0.4 ml glutardialdehyde (25% w/v) to
80 ml of the sensitizing solution
Silver solution Silver nitrate solution (2.5% w/v) 25 ml
Water to 250 ml
Developing solution Sodium carbonate (6.25 g) 1 packet
Water to 250 ml
Stir vigorously to dissolve the sodium carbonate
Before use: Add 0.05 ml formaldehyde (37% w/v) to 80 ml
of the developing solution
Stop solution Glacial acetic acid 12.5 ml
Water to 250 ml
Washing solution Water

20 Instructions 71-7177-00 AL
 Staining procedure
Steps to be programmed into the development method file:
.

Step Time Temp

Solution IN OUT (min.) (°C)
1 Fixing solution TCA 1 91 5 20
2 Fixing solution 10% Ethanol 2 0 4 50
3 Fixing solution 10% Ethanol 2 0 4 50
4 Sensitizing solution 3 91 6 50
5 Fixing solution 10% Ethanol 2 0 3 50
6 Fixing solution 10% Ethanol 2 0 5 50
7 Water 4 0 2 50
8 Water 4 0 2 50
9 Silver solution 5 91 10 40
10 Water 4 0 0.5 20
11 Water 4 0 0.5 20
12 Developing solution 6 91 1 to 52 20
13 Stop solution 7 0 10 50
1
Please consult local regulations for information on proper disposal
2
Guideline only, need to be optimized

Instructions 71-7177-00 AL 21
4 Troubleshooting guide
Problem Cause Remedy
Bands / spots do not develop Temperature of solution too low Keep temperature of solution
between 5 and 25 °C
Gel not washed thoroughly Repeat washing procedure
enough
Did not fix properly Check fixing solution or fixing
properties of your particular
protein(s)
Background is excessively dark Temperature of developing Chill the developing solution in
(over-developed) solution too high the refrigerator before use
Water impure Use deionized water whenever
contaminated possible
The carrier ampholytes have not After stop solution step, put the
been washed out gel in 1% sodium thiosulphate
solution for several hours to
reduce the silver stain
Bands lighter than the Overloading Lower the amount of protein
background loaded onto the gel
Smearing or blackening in lanes Overloading Dilute samples (remember silver
where protein loaded staining is approximately 50 to
100 times more sensitive than
Coomassie Blue)
Silver mirror reaction Insufficient washing with water Extend the time of washing after
staining
Dirty tray Replace the chamber for
development with a new clean
one
Insufficient shaking During the development step
shake the tray sufficiently to
prevent the gel from sticking to
the bottom of the tray

22 Instructions 71-7177-00 AL
5 Ordering information
5.1 PlusOne Silver Staining Kit, Protein
Product Code No.
PlusOne Silver Staining Kit, Protein 17-1150-01

5.2 Chemicals and accessories

Chemicals Code No.
PlusOne Glycerol 17-1325-01
PlusOne Urea 17-1319-01
PlusOne Iodoacetamide RPN6302
PlusOne CHAPS 17-1314-01
PlusOne Triton X-100 17-1315-01
PlusOne Acrylamide PAGE 17-1303-01
PlusOne N,N'-Methylene-bisacryalmide 17-1306-01
PlusOne Ammonium Persulphate 17-1311-01
PlusOne TEMED 17-1312-01
PlusOne Bind-Silane (25 ml) 17-1330-01
PlusOne Tris 17-1321-01
PlusOne Glycine 17-1323-01
PlusOne Tween 20 17-1316-01
PlusOne SDS 17-1313-01
PlusOne DTT 17-1318-01
PlusOne Bromophenol blue (BFB) 17-1329-01
2D Quant Kit 80-6483-56
DeStreak™ Rehydration Solution 17-6003-19
DeStreak Reagent 17-6003-18

Accessories Code No.

Staining Tray Set 80-6468-17
Cellophane Sheets 80-1129-38
Gel frame, large (up to 21 × 26 cm gels) 80-6429-22
Gel loading platform large 80-6429-41
(works with 80-6429-22)

Instructions 71-7177-00 AL 23
5.3 Precast gels and related products
Molecular weight and pI markers
Product Code No.
Peptide Marker Kit (MW range 2 512 to 16 949) 80-1129-83
LMW-SDS Marker Kit (MW range 14 000 to 97 000) 17-0446-01
HMW-SDS Marker Kit (MW range 53 000 to 220 000) 17-0615-01
HMW Native Marker Kit (MW range 66 000 to 669 000) 17-0445-01
Broad pI Kit pH 3 to 10 17-0471-01
Low pI Kit pH 2.5 to 6.5 17-0472-01
High pI Kit pH 5 to 10.5 17-0473-01

Precast gels and buffer strips for SDS PAGE, IEF and Native PAGE
Product Code No.
ExcelGel SDS Homogeneous 7.5 80-1260-01
ExcelGel SDS Homogeneous 12.5 80-1261-01
ExcelGel SDS Homogeneous 15 80-1262-01
ExcelGel SDS, gradient 8-18 80-1255-53
ExcelGel XL SDS 12-14 17-1236-01
ExcelGel SDS Buffer Strips 17-1342-01
CleanGel IEF 18-1031-54
Immobiline DryPlate pH 4.0 to 7.0 80-1128-28
Immobiline DryPlate pH 4.2 to 4.9 80-1128-29
Immobiline DryPlate pH 4.5 to 5.4 80-1128-30
Immobiline DryPlate pH 5.0 to 6.0 80-1128-31
Immobiline DryPlate pH 5.6 to 6.6 80-1128-32
PhastGel Gradient 4 to 15 17-0678-01
PhastGel Gradient 10 to 15 17-0678-01
PhastGel Gradient 8 to 25 17-0542-01
PhastGel Homogeneous 7.5 17-0622-01
PhastGel Homogeneous 12.5 17-0623-01
PhastGel Homogeneous 20 17-0624-01
PhastGel Homogeneous High Density 17-0679-01
PhastGel IEF - 3 to 9 17-0543-01
PhastGel IEF - 4 to 6.5 17-0544-01
PhastGel IEF - 5 to 8 17-0545-01
PhastGel Dry 17-0677-01
PhastGel Buffer Strips SDS17-0516-01
PhastGel Buffer Strips Native 17-0517-01

24 Instructions 71-7177-00 AL
 2-D, First dimension
Immobiline Code No. Immobiline Code No.
DryStrip, 7 cm DryStrip, 11 cm
pH 3 to 5.6 NL 17-6003-53 pH 3 to 5.6 NL 17-6003-54
pH 5.3 to 6.5 17-6003-58 pH 5.3 to 6.5 17-6003-59
pH 6.2 to 7.5 17-6003-63 pH 6.2 to 7.5 17-6003-64
pH 7 to 11 NL 17-6003-68 pH 7 to 11 NL 17-6003-69
pH 3 to 11 NL 17-6003-73 pH 3 to 11 NL 17-6003-74
pH 4 to 7 17-6001-10 pH 4 to 7 18-1016-60
pH 3 to 10 17-6001-11 pH 3 to 10 18-1016-61
pH 3 to 10 NL 17-6001-12 pH 6 to 11 17-6001-95
pH 6 to 11 17-6001-94

Immobiline Code No. Immobiline Code No.

DryStrip, 13 cm DryStrip, 18 cm
pH 3 to 5.6 NL 17-6003-55 pH 3 to 5.6 NL 17-6003-56
pH 5.3 to 6.5 17-6003-60 pH 5.3 to 6.5 17-6003-61
pH 6.2 to 7.5 17-6003-65 pH 6.2 to 7.5 17-6003-66
pH 7 to 11 NL 17-6003-70 pH 7 to 11 NL 17-6003-71
pH 3 to 11 NL 17-6003-75 pH 3 to 11 NL 17-6003-76
pH 4 to 7 17-6001-13 pH 4 to 7 17-1233-01
pH 3 to 10 17-6001-14 pH 3 to 10 17-1234-01
pH 3 to 10 NL 17-6001-15 pH 3 to 10 NL 17-1235-01
pH 6 to 11 17-6001-96 pH 3.5 to 4.5 17-6001-83
pH 4.0 to 5.0 17-6001-84
pH 4.5 to 5.5 17-6001-85
pH 5.0 to 6.0 17-6001-86
pH 5.5 to 6.7 17-6001-87
pH 6 to 9 17-6001-88
pH 6 to 11 17-6001-97

Instructions 71-7177-00 AL 25
 Immobiline Code No. IPG buffers Code No.
DryStrip, 24 cm
pH 3 to 5.6 NL 17-6003-57 IPG Buffer pH 3.5 to 5.0 17-6002-02
pH 5.3 to 6.5 17-6003-62 IPG Buffer pH 4.5 to 5.5 17-6002-04
pH 6.2 to 7.5 17-6003-67 IPG Buffer pH 5.0 to 6.0 17-6002-05
pH 7 to 11 NL 17-6003-72 IPG Buffer pH 5.5 to 6.7 17-6002-06
pH 3 to 11 NL 17-6003-77 IPG Buffer pH 4 to 7 17-6000-86
pH 3.5 to 4.5 17-6002-38 IPG Buffer pH 6 to 11 17-6001-78
pH 4.0 to 5.0 17-6002-39 IPG Buffer pH 7 to 11 NL 17-6004-39
pH 4.5 to 5.5 17-6002-40 IPG Buffer pH 3 to 10 NL 17-6000-88
pH 5.0 to 6.0 17-6002-41 IPG Buffer pH 3 to 10 17-6000-87
pH 5.5 to 6.7 17-6002-42 IPG Buffer pH 3 to 11 NL 17-6004-40
pH 3 to 7 NL 17-6002-43
pH 3 to 10 17-6002-44
pH 3 to 10 NL 17-6002-45
pH 4 to 7 17-6002-46
pH 6 to 9 17-6001-88

2-D, Second dimension

Product Code No.
ExcelGel SDS, gradient 8-18 80-1255-53
ExcelGel XL SDS 12-14 17-1236-01
ExcelGel SDS Buffer Strips 17-1342-01
DALT Gel 12.5 17-6002-36
DALT Gel Buffer Kit 17-6002-36

5.4 Electrophoresis equipment

Product Code No.
Ettan DALT Twelve Separation Unit 80-6466-46
Ettan DALTsix Large Vertical System 80-6485-08
SE 600 Ruby Vertical Unit 80-6479-57
SE 400 Vertical Unit 80-6155-24
Multiphor™ II Electrophoresis System 18-1018-06
MultiTemp™ III Thermostatic Circulator 18-1102-78
PhastSystem™ 18-1018-23
miniVE Vertical Electrophoresis System 80-6418-77
Mini-Vertical Units 80-6147-45

26 Instructions 71-7177-00 AL
Instructions 71-7177-00 AL 27
For contact information for your GE tagline and GE monogram are trademarks of General Electric
Company.
local office, please visit Ampholine, CleanGel, DeStreak, Ettan, ExcelGel, Immobiline,
www.gelifesciences.com/contact Multiphor, MultiTemp, PhastGel and PhastSystem are trademarks of
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All third party trademarks are the property of their respective owners.
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Björkgatan 30 First published Oct. 1993.

751 84 Uppsala All goods and services are sold subject to the terms and conditions of
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your local GE Healthcare representative for the most current
information.

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71-7177-00 AL 09/2008