Bio-Rad Silver Stains User Manual

Dodeca™Silver Stain Kit

Instruction Manual

For Technical Service, Call Your Local Bio-Rad Office

or in the US, Call 1-800-4BIORAD (1-800-424-6723)

Catalog #161-0480
Catalog #161-0481

Table of Contents

Section 1 Introduction........................................................................................................................ 1

Section 2 Specifications .................................................................................................................... 2

Section 3 Reagent Preparation .......................................................................................................... 4

Section 4 Polyacrylamide Gel Staining Procedure .............................................................................. 9

Section 5 Troubleshooting Guide ...................................................................................................... 10

Section 6 Product Information .......................................................................................................... 13

Section 1
Introduction

The Dodeca™silver stain kit is an easy-to-use kit for the detection of nanogram levels of proteins in
polyacrylamide gels. The kit is formulated with concentrated aqueous solutions that enable the user to
quickly prepare volumes of working solutions that may be easily tailored to the number of gels to be stained.
Furthermore, the kit is designed to be compatible with the high-throughput Dodeca stainer by facilitating
reagent preparation and instrument cleaning.

The Dodeca silver stain kit is based on the method described by Sinha et al. (2001) in which protein-bound
silver ions are chemically reduced to form visible metallic silver. Additionally, no protein-modifying
glutaraldehyde is used, and only minimal concentrations of the reducing agent, formaldehyde, which is
required for silver stain development. Consequently, this procedure yields silver-stained proteins that are
compatible with mass spectrometry for protein identification analysis.

Reference

Sinha P et al., A new silver staining apparatus and procedure for matrix-assisted laser desorption/ionization-time of flight analysis of proteins
after two dimensional electrophoresis, Proteomics 1, 835–840 (2001)

1

Section 2
Specifications

Catalog #

161-0481 161-0480

Maximum Dodeca stainer volume 7 L 10 L

Component solutions

Sensitizer concentrate (SC) 700 ml 1 L

Background reducer concentrate (BRC) 75 ml 125 ml

Silver reagent concentrate (SRC) 140 ml 200 ml

Development buffer concentrate (DBC) 700 ml 1 L

Image developer concentrate (IDC)
(contains formaldehyde) 2.25 ml 2.25 ml

Quick reference card 1 each 1 each

Kit storage temperature Ambient

Optimum kit operating temperature 21–25°C

Bovine serum albumin (BSA) staining sensitivity ≥0.5 ng

Shelf life 12 months

2

Table 1. Recommended Dodeca silver stain kit and Dodeca stainer for various gel sizes.

Kit Size Stainer Size Tray Size Gel Sizes (W x L) Bio-Rad Gel Formats

Dodeca silver stain kit, large Large Dodeca stainer Large staining tray 25.6 x 23 cm PROTEAN

®

Plus precast gels

(161-0480) (165-3400) 25 x 20.5 cm PROTEAN Plus handcast gels

(require one attachment per
tray, catalog #165-3417)

Dodeca silver stain kit, small Small Dodeca stainer Small staining tray 20 x 20.5 cm PROTEAN Plus handcast gels

(161-0481) (165-3401) 18 x 20 cm PROTEAN

®

II XL handcast

and precast gels

16 x 20 cm PROTEAN II xi handcast gels

16 x 16 cm PROTEAN II xi handcast

and precast gels

13.3 x 8.7 cm Criterion

™

gels (up to 24 gels,
require one attachment per
tray, catalog #165-3418)

3

Section 3
Reagent Preparation

Additional Materials Needed

In addition to the components in the Dodeca silver stain kit, the following materials are required:

• Reagent-grade ethanol and glacial acetic acid

• Deionized, distilled, or high purity water. Water of ≥18 MΩ-cm resistivity is recommended

• Dodeca stainer (catalog #165-3400 or 165-3401), or glass or plastic staining trays

• When not using a Dodeca stainer, all steps in the procedure require agitation of the gel in solution — for
best results, a shaker table is recommended

Warnings and Precautions

Read each label in the kit before beginning the procedure.

Always wear powder-free laboratory gloves when handling gels to prevent protein contamination.

The image developer concentrate contains formaldehyde and should be used in properly ventilated areas.
Avoid contact with skin. In case of contact with eyes, flush with copious amounts of water and contact a
physician.

See Material Safety Data Sheets for additional information about each solution.

Dispose of waste material according to local regulations.

4

Working Solution Volumes

For best results, prepare each working solution no more than 24 hr before use. Add the image developer
concentrate to the developing working solution within 15 minutes of use. Note: The volume corresponding

to water (Table 2, 3 and 4) indicates the final volume of the working solution.

Please refer to the appropriate section below to determine the proper working solution volumes for staining

1) 12 PROTEAN gels in a Dodeca stainer, 2) fewer than 12 gels in a Dodeca stainer, or 3) individual gels in a
user-provided staining container.

1) Dodeca stainer with 12 trays. When using the full capacity of the Dodeca stainer, prepare each
working solution as directed by Table 2. Please note that the cover tray is not counted.

Table 2. Dodeca stainer solution volumes.

Working Solutions Reagents Small Dodeca Stainer Large Dodeca Stainer

12 Trays 12 Trays

Fixing Ethanol 2.8 L 4.0 L

Glacial acetic acid 700 ml 1.0 L
Water To 7 L To 10 L

Sensitizing SC (10×) 700 ml 1.0 L

BRC (100×) 70 ml 100 ml
Ethanol 2.1 L 3.0 L
Water To 7 L To 10 L

Staining SRC (50×) 140 ml 200 ml

Water To 7 L To 10 L

Developing DBC (10×) 700 ml 1.0 L

IDC (5,000×) 1.4 ml 2.0 ml
BRC (20,000×) 350 µl 500 µl
Water To 7 L To 10 L

Stopping Glacial acetic acid 350 ml 500 ml

Water To 7 L To 10 L

5

2) Dodeca stainer with less than 12 trays. Although the provided volumes of the concentrates are

optimized to stain 12 PROTEAN gels (or 24 Criterion gels) in a Dodeca stainer, fewer gels also may be
stained. However, because of inherent dead volumes in the Dodeca stainer (as with most instruments),
a slightly higher solution volume per tray ratio is required for optimal results. Using Table 3, the required
working solution volumes are calculated by taking the number of staining trays used (not including the
cover tray) multiplied by the volume listed in the appropriate “Per Tray” column, and then adding the
corresponding volume listed under the adjacent “Plus” column. For example, six PROTEAN Plus gels
stained using six trays in the large Dodeca stainer require (6 trays x 750 ml) + 1.5 L, for a total volume of

6.0 L for each working solution. Silver staining with fewer than four trays is not recommended.

6

Table 3. Dodeca stainer volumes for less than 12 trays.*

Working Solutions Reagents Small Dodeca Stainer Large Dodeca Stainer

4–11 Trays 4–11 Trays

Per Tray Plus Per Tray Plus

Fixing Ethanol 200 ml 400 ml 300 ml 600 ml

Glacial acetic acid 50 ml 100 ml 75 ml 150 ml
Water To 500 ml To 1 L To 750 ml To 1.5 L

Sensitizing SC (10×) 50 ml 100 ml 75 ml 150 ml

BRC (100×) 5 ml 10 ml 7.5 ml 150 ml
Ethanol 150 ml 300 ml 225 ml 450 ml
Water To 500 ml To 1 L To 750 ml To 1.5 L

Staining SRC (50×) 10 ml 20 ml 15 ml 30 ml

Water To 500 ml To 1 L To 750 ml To 1.5 L

Developing DBC (10×) 50 ml 100 ml 75 ml 150 ml

IDC (5,000×) 100 µl 200 µl 150 µl 300 µl
BRC (20,000×) 25 µl 50 µl 37.5 µl 75 µl
Water To 500 ml To 1 L To 750 ml To 1.5 L

Stopping Glacial acetic acid 25 ml 50 ml 37.5 ml 75 ml

Water To 500 ml To 1 L To 750 ml To 1.5 L

*Please refer to page 6 for further details

7

3) Individual gels. Prepare working solutions according to Table 4. To stain multiple gels, multiply the

solution volumes by the appropriate number of gels. The working solution volumes listed below are
recommended volumes — the actual required volume may vary depending on the dimensions of the
staining container used. For all cases, sufficient volumes of working solution should be prepared to
completely submerge the gel to be stained.

Table 4. Suggested working solution volumes for individual gels.

Working Solutions Reagents Individual Mini or Individual Large Gels

Criterion

™

Gels 16 x 16 cm or larger

(250 ml) (1 L)

Fixing Ethanol 100 ml 400 ml

Glacial acetic acid 25 ml 100 ml
Water To 250 ml To 1 L

Sensitizing SC (10×) 25 ml 100 ml

BRC (100×) 2.5 ml 10.0 ml
Ethanol 75 ml 300 ml
Water To 250 ml To 1 L

Staining SRC (50×) 5 ml 20 ml

Water To 250 ml To 1 L

Developing DBC (10×) 25 ml 100 ml

IDC (5,000×) 50.0 µl 200.0 µl
BRC (20,000×) 12.5 µl 50.0 µl
Water To 250 ml To 1 L

Stopping Glacial acetic acid 12.5 ml 50.0 ml

Water To 250 ml To 1 L

8

Section 4
Polyacrylamide Gel Staining Procedure

Before starting this procedure, prepare the working solutions as described on pages 5–8.

Step Description Small Gels Large Gels

(Mini or Criterion (16 × 16 cm
Gels) or larger)

1 Fixing. Place gel(s) in fixing solution >30 min >1 hr

and shake for indicated period of time. (overnight acceptable) (overnight acceptable)

2 Sensitizing. Remove the fixing solution 30 min 30 min

and shake gel(s) in the sensitizing solution.

3 Washing. Remove sensitizing solution 3 × 5 min 3 × 10 min

and wash 3 times with one working
volume of water.

4 Staining. Remove water and shake 20 min 30 min

in staining solution.

5 Rinsing. Remove staining solution 1 × 1 min, (2 × 1 min)

†

1 × 1 min, (2 × 1 min)

†

and rinse the gel(s) in one working
volume of water.

6 Developing. Remove water and shake gel(s) 10–30 min 10–30 min

in developing solution.

7 Stopping. Remove developing solution and 10 min 10 min

shake gel(s) in stopping solution.

8 Washing. Wash gels in one working volume 10 min 10 min

of water. Gel(s) may be dried, imaged, or used
to excise visualized proteins.

†

When using the Dodeca stainer, pay special attention to minimize the solution draining and filling time between steps 5 and 6, and between

steps 6 and 7. However, gels stained individually should be rinsed in step 6 with two 1 min water washes.

9

10

Section 5
Troubleshooting Guide

Problem Cause Solution

Background dark or uneven Agitation too gentle. Increase agitation speed during

gel incubation and ensure gel is
completely submerged.

Dodeca stainer or staining Thoroughly clean Dodeca stainer
containers contain residue from or containers with soap and
prior cleaning or staining runs. water. Rinse only glass

containers with 50% nitric
acid, followed by thorough
water rinse to remove the acid.

Fingerprints or powder from Use powder-free laboratory
gloves cause staining artifacts. gloves.

Interfering components (glycine, Increase fixing time or use
SDS, etc.) not completely multiple fixing cycles to
removed from gel. completely wash interfering

compounds from gel.

Sensitizing working solution Prepare new sensitizing
improperly prepared. solution with BRC.

Problem Cause Solution

Background dark or uneven (cont.) Contaminants in water. Prepare all solutions with

double-distilled water with
a resistance of at least
18 MΩ-cm.

Low protein staining sensitivity or Excessive silver ions washed out Verify that each post­no staining of gel. staining water wash is

exactly 1 min long.

pH of developing working Verify the working solution
solution too low. pH = 11.6 ± 0.5, and

prepare new developing
solution with development
buffer concentrate, if
necessary.

Reducer not present in Prepare new developing
developing working solution. solution with image

developer concentrate.

Insufficient fixing due to variable Extend the time of the
fixing characteristics of different fixing step.
proteins.

11

Problem Cause Solution

Low protein staining sensitivity or Development terminated Increase the development
no staining (cont.) prematurely. time. Protein stain may be

developed up to 30 min
with a minimal increase
in background.

Negative protein staining (light bands Protein sample overloaded. Reduce the amount of
or spots on a darker background) protein loaded.

12

Section 6
Product Information

Catalog # Description

161-0480 Dodeca Silver Stain Kit, large (10 L), solutions provided in volumes optimized for

the large Dodeca stainer

161-0481 Dodeca Silver Stain Kit, small (7 L), solutions provided in volumes optimized for

the small Dodeca stainer
161-0472 Image Developer Concentrate, 2.5 ml
161-0473 Sensitizer Concentrate, 700 ml
161-0474 Sensitizer Concentrate, 1 L
161-0475 Background Reducer Concentrate, 75 ml
161-0476 Background Reducer Concentrate, 125 ml
161-0477 Silver Reagent Concentrate, 140 ml
161-0478 Silver Reagent Concentrate, 200 ml
161-0479 Development Buffer Concentrate, 700 ml
161-0482 Development Buffer Concentrate, 1 L
165-3400 Dodeca Stainer, large
165-3401 Dodeca Stainer, small
165-3403 Dodeca Stainer and Dodeca Silver Stain Kit, large
165-3404 Dodeca Stainer and Dodeca Silver Stain Kit, small
345-9920 Criterion Staining/Blotting Trays, 10 trays

13

Catalog #
161-0480
161-0481

4110150 Rev B