65
Additional Staining Protocols,
Continued
Pro-Q
®
Sapphire
532 Oligohistidine
Gel Stain
Instructions for specific staining His-tagged proteins on E-PAGE
™
Gels using
Pro-Q
®
Sapphire 532 Oligohistidine Gel Stain is described in this section.
The total staining time is ~5 hours.
Due to the thickness of E-PAGE
™
Gels, the Pro-Q
®
Sapphire 532 Oligohistidine
Gel Stain
may not be optimally sensitive
for your particular staining
application.
To visualize His-tagged proteins on E-PAGE
Gels, we
recommend the InVision
™
His-tag In-Gel Stain (page 48).
Materials Needed
You will need the following items for staining one E-PAGE
Gel:
•
Pro-Q
®
Sapphire 532 Oligohistidine Gel Stain
•
Fixing solution (50% ethanol, 10% acetic acid, made with deionized water)
•
Deionized water
•
Microwave safe
staining tray
•
Rotary shaker
•
Microwave oven (700–1000 watts)
•
UV transilluminator equipped with a standard camera or laser scanners
Oligohistidine
Staining
Procedure
For all steps described below, be sure to use sufficient volume of reagent to
completely cover the gel using a suitable container such that the gel moves freely.
1.
After electrophoresis, remove the gel from the cassette (page 24) and place the
gel in a clean
microwave safe
staining container.
2.
Fix the gel in fixing solution (50% ethanol, 10% acetic acid in deionized water)
for 45 minutes at room temperature with gentle shaking. Decant the fixing
solution and add fresh fixing solution to the container and repeat the fixing
step again for 45 minutes at room temperature. Decant the fixing solution.
3.
Add deionized water to the gel and microwave at high power for 45 seconds.
Remove the gel from the microwave and wash the gel for 10 minutes at room
temperature with gentle shaking. Decant the water and repeat the wash step
for another 10 minutes again with deionized water. Decant the water.
4.
Stain the gel with Pro-Q
®
Sapphire 532 Oligohistidine Gel Stain. Incubate the
gel in the
DARK
for 30–60 minutes at room temperature with gentle shaking.
Decant the stain.
5.
Wash the gel with deionized water twice for 1 hour each time at room
temperature with gentle shaking. You may wash the gel overnight to improve
sensitivity and to obtain clear background.
Summary of Contents for E-PAGE Gels
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