Bio-Rad Negative Stains - Manual (Page 5)

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.
2
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.)
5
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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