Bio-Rad Negative Stains User Manual

Copper Stain & Destain Kit

for Electrophoresis

Instruction Manual

Catalog Number

161-0470

Table of Contents

Page

Section 1 Introduction ……………………………………… 1

1.1 Introduction and Principle ………………………………… 1

1.2 Product Description ……………………………………… 2

1.3 Materials Required But Not Supplied …………………… 3

1.4 Safety Considerations……………………………………… 3

1.5 Disposal Methods ………………………………………… 3

Section 2 Instructions………………………………………… 4

2.1 Copper Stain Protocol …………………………………… 4

2.2 Copper Destain Protocol ………………………………… 5

Section 3 Additional Information…………………………… 7

3.1 Commonly Asked Questions ……………………………… 7

3.2 References ………………………………………………… 8

Section 4 Product Information ……………………………… 9

4.1 Copper Stain ……………………………………………… 9

4.2 Related Materials ………………………………………… 9

Section 1
Introduction

1.1 Introduction and Principle

The Copper Stain & Destain Kit for Electrophoresis provides a rapid,
reversible visualization of protein bands on a Laemmli SDS-PAGE gel.
The Copper Stain & Destain Kit is modified from a new staining method
developed by Lee et al.
from the gel

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and subsequently used for blotting,3as antigens in ELISA,

for amino acid sequencing,5or for other analyses.

The Copper Stain & Destain Kit offers several advantages over the
traditional methods of Coomassie Brilliant Blue (CBB) or silver staining.
First, the Copper Stain & Destain procedures save time. For a typical

0.75 mm gel, a brief water rinse is followed by a 5 minute incubation
with Copper Stain. The gel is destained within 15 minutes, so this entire
process can be completed in less than 30 minutes. Second, the Copper
Stain & Destain procedures are convenient. Only a simple dilution of the
reagents is required. In addition, the proteins are reversibly fixed within
the gel, so that the polyacrylamide gel can be destained and the proteins
eluted for further study.

The Copper Stain, a negative stain, produces a blue-green opaque
background. The protein bands are visualized against a black surface, and
can be photographed to provide a permanent record, as shown in Figure 1.

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It enables the proteins to be quantitatively eluted

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®

Fig. 1. Mini-PROTEAN

Rad's LMW SDS-PAGE standards were diluted and electrophoresed at 200 V

for 30 minutes. Standards were serially diluted from 500 ng of each protein per

well to 3.91 ng of each protein per well. The gel was subsequently stained with

Copper Stain.

II 12% ready gel stained with Copper Stain. Bio-

The Copper Stain can be ordered separately, as catalog number 161-

0471. The Copper Destain, a 10x Tris/Glycine buffer, can be reordered
in a 1 liter bottle as catalog number 161-0734.

The Copper Staining procedure is recommended for mini-gels,
especially those of 1.0 mm thickness or less. The quantities included
with this kit can be used to stain 25 mini-gels, using 50 ml Copper Stain
for each gel, or 2 full-size gels, using about 600 ml stain for each gel.

1.3 Materials Required But Not Supplied

Graduated cylinders

Distilled, deionized water

Staining and rinsing containers. To optimize the number of gels

stained per kit, small staining containers, such as the lid of a pipet tip
box, should be used for staining mini-gels.

The gel can be stored in water for several weeks without loss of

resolution of the protein bands.

1.2 Product Description

The Copper Stain & Destain Kit, catalog number 161-0470, contains:

125 ml Copper Stain, 10x
125 ml Copper Destain, 10x
Laminated instruction card
Complete instruction manual

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1.4 Safety Considerations

Eye protection and gloves should be worn while handling this
product.

1.5 Disposal Methods

Laws governing disposal of laboratory chemicals vary by region.
Check local laws for disposal of Copper Stain.

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Section 2
Instructions

2.1 Copper Stain Protocol

1. Dilute one part Copper Stain with nine parts water to make the
working reagent. Alternatively, the entire contents of the bottle can
be emptied into a 1 liter bottle containing 900 ml of DDI water. Mix
the solution thoroughly.

2. Remove the gel from the electrophoresis cell.

3. Place the gel in a container with distilled, deionized water. See
Table 1 for recommended rinse time. Place the container on an
orbital mixing platform and set to a low mix speed.

4. Transfer the gel to diluted Copper Stain. Completely immerse the gel
to insure even staining. (A Mini-PROTEAN II gel can be completely
immersed in as little as 50 ml of Copper Stain, provided the staining
vessel is small.) Allow 5 minutes for the gel to develop.

5. Transfer the gel to a container filled with DDI water and rinse for 3
minutes. Discard this water wash and replace it with fresh DDI
water. The gel can be stored for weeks in water.

6. To visualize the protein bands, place the gel against a black
background. (The reverse side of the laminated instruction card is
colored black for this purpose.) The protein bands will be visible as
dark bands against an opaque blue-green background. The gel can

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now be photographed to provide a permanent record of the
separation. For best results, illuminate the gel at an oblique angle
with four high-intensity 150-W flood light bulbs. The optimum angle
of exposure is empirical.

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Table 1. Copper Staining Wash Times

The following are recommended water rinse times done prior to
copper staining. The optimum time is noted with an asterisk, although
any time within the range can be used.

Gel Thickness Rinse Time

0.5 mm 15*-30 seconds

0.75 mm 30*-60 seconds

1.0 mm 3*-5 minutes

2.2 Copper Destain Protocol

1. Consult Table 2 for Copper Destain dilutions and wash times. Note
that 1.5 mm gels are not recommended for this procedure; the rinse
times are long, thereby increasing the likelihood of band spreading.

2. Completely immerse the stained gel in a 1:10 dilution of Copper
Destain, and gently agitate for 5 minutes. (Mini-gels require 100 ml of
destaining solution for each step. Full size gels require considerably
more destain solution. Volumes must be determined empirically.)

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3. Replace this rinse with fresh, diluted Copper Destain and gently
agitate again for the recommended time. Repeat with 1:20 Copper
Destain, if recommended in Table 2.

4. The gel is now ready for Coomassie staining, silver staining,
blotting, or other analyses.

Table 2. Copper Destaining Wash Times

The following are recommended destaining wash times. The
destaining procedure has been optimized for mini-gels. Slight
variations in the protocol may be necessary to optimize results with
full-size gels.

Gel Thickness Destain #1 Destain #2 Destain #3

0.5 mm 5 minutes 3 minutes ——

0.75 mm 5 minutes 5 minutes 5 minutes

1.0 mm 5 minutes 10 minutes 5 minutes

The Copper Destain solution is diluted 1:10 with water. If necessary,

Destain #3 can be further diluted to 1:20, which will help to conserve
reagents.

Section 3
Additional Information

3.1 Commonly Asked Questions

1. Can I blot the copper Yes, after destaining. The Copper
stained gel? Stain reversibly fixes the proteins in

the gel.

2. Am I able to stain the gel Yes. In fact, only with a silver stain
later with either CBB must you first destain the gel. The
or silver? low pH of the Coomassie staining

solution acts as a destainer.

3. Can the Copper Stain be For optimum results, discard the
reused? solution after each use. Results show

that it might work a second time, but
with each use the staining is less
effective.

4. Can the Copper Destain be No, this is not recommended.
reused?

5. Can I stain DNA in a Bands do not develop when copper
TBE gel? staining a DNA gel.

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6. Can a native gel be stained Yes, but the staining pattern is
with Copper Stain? reversed. In this case, the protein

bands stain an opaque blue-green
while the background remains clear.
However, the effect of staining native
proteins has not been examined.

7. How many gels can be This is dependent on the size of the
stained per kit? staining container. A mini-gel can be

stained in as little as 50 ml of Copper
Stain, providing that the entire gel is
submerged during the staining step.
The limiting factor is the volume of
the stain, not the concentration of
Copper Stain.

3.2 References

1. Lee, C., Levin, A. and Branton, D., Anal. Biochem., 166, 308 (1987).

2. Dzandu, J. K., Johnson, J. F. and Wise, G. E., Anal. Biochem., 174, 157 (1988).

3. Wang, D., Dzandu, J. K., Hussain, M. and Johnson, R. M., Anal. Biochem., 180,

311 (1989).

4. Vos, G. J. and Gardiner, P.R., Parasitology, 100, 93 (1990).

5. Vanfleteren, J. R. and Peeters, K., J. Biochem. Biophys. Methods, 20, 227 (1990).

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Section 4
Product Information

4.1 Copper Stain

161-0470 Copper Stain & Destain Kit for Electrophoresis, includes 125 ml

10x Copper Stain, 125 ml 10x Copper Destain, laminated protocol,

and instruction manual
161-0471 Copper Stain, 10x, 125 ml
161-0734 10x Tris/Glycine (Copper destain), 10x, 1L

4.2 Related Materials

161-0900 Mini-PROTEAN II Ready Gels, 7.5% single percentage gels,

0.375 M Tris-HCl, 10

161-0901 Mini-PROTEAN II Ready Gels, 12% single percentage gels, 0.375

M Tris-HCl, 10
161-0902 Mini-PROTEAN II Ready Gels, 4 - 15% gradient gels, 0.375 M

Tris-HCl, 10
161-0903 Mini-PROTEAN II Ready Gels, 4 - 20% gradient gels, 0.375 M

Tris-HCl, 10
161-0732 10x Tris/Glycine/SDS, 1L
161-0755 10x Tris/Glycine/SDS, 6 x 1L
161-0757 10x Tris/Glycine, 6 x 1L

For information on the vertical slab cell systems, PROTEAN II xi cells
and the Mini-PROTEAN II cells, consult the catalog or contact your
local Bio-Rad representative.

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Bio-Rad Laboratories, 2000 Alfred Nobel Drive, Hercules, CA 94547

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LIT464 Rev B