C.B.S. Scientific TTGE-2401 - Manual (Page 5)

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GEL STAINING
These composite polymer gels are very strong and change shape minimally when placed in different
solutions. However, the composite polymer tends to hold SDS strongly compared to other 1mm thick
mini-gels, so a pre-wash or extra staining time is necessary to remove the SDS which competes with the
proteins for the stain in more sensitive staining methods. This step is NOT necessary when using the
ClearPAGE™ Instant Blue Stain.
1) Staining with ClearPAGE™ Instant Blue Stain
a) BEFORE USE: Mix the Instant Blue solution immediately before use by
gently inverting the bottle a few times (do not shake the bottle to mix the
solution). One may rinse the gel surfaces briefly with ultrapure water, but
do NOT wash the gel, as standing in water before staining will reduce band
sharpness. Fixing is also NOT recommended.
b) GEL STAINING: Place each gel in a separate small container (lids for
yellow pipette tips work well). Use the following amount of stain for
different size gels: Use 25ml per gel for gels approximately 8 x 8 cm (from
10 x 10 cm cassettes, such as ClearPAGE or Invitrogen Gels). Use 20 ml per
gel for gels approximately 6 x 8cm (from 8 x 10cm cassettes, such as BIO-
RAD, Precise or Gradipore gels) Larger Volumes are required for larger gels
and for larger containers.
c) Shake gently until desired band intensity is achieved. Generally, 50ng or
less can be seen in 10 minutes. Band intensity reaches a maximum in 30 to
60 minutes depending on gel thickness.
d) Destaining does NOT improve sensitivity, but the gels may be washed in
ultrapure water for 15 minutes to remove the free stain from the gel and get
clear backgrounds. The gel may be stored in ultrapure water.
2) Staining with Coomassie™ Brilliant Blue R-250
When using R-250 stain solutions (40% ethanol/10% acetic acid/0.1% Brilliant Blue R-250), follow
standard protocols or use the following recommended by ClearPAGE. Use 20% ethanol/5% acetic acid
as the R250 destain solution, half the usual destain concentration, combined with an absorbent paper
such as paper towel or lab wipes, which will prevent the proteins from destaining.
STEP Heated Protocol Ambient Temperature Protocol
Stain Rock with 50ml warm (50-65ºC) stain 15 min. Rock with 50ml R250 stain 1 to 2 hours
Destain Rock with 50ml warm R250 destain with paper
towels or lab wipes for 1 to 24 hrs
Rock with 50ml R250 destain 3 to 24 hrs.
3) Staining with “Safe” Stains (Coomassie G250-based stains)
For use with Simply Blue™ and Gel Code™ Blue or Bio-Safe™ stains:
STEP Heated Protocol Ambient Temperature Protocol
Fix and
Wash
Heat 20% Ethanol at 50º-70º. Rock 5 minutes,
50ml/gel You must do this step.
Rock 30 minutes with 50ml 20% Ethanol per gel
Water
Wash
Heat water to circa 80ºC. Rock 2 minutes,
100ml/gel. Repeat once. Do not use tempera-
tures higher than 80ºC.
Rock 5 minutes with 100ml water per gel. Repeat twice.
Stain Heat stain to 70º-80ºC. Rock 10 minutes,
25ml/gel
Use 25ml stain per gel. Rock 1 hour.
Destain Heat 10% Ethanol (50ml/gel) at 50º-70ºC.
Rock 15 minutes - 16 hours with adsorbent
paper
Use 100ml 10% ethanol per gel. Rock 2 to 16 hours.
Storage Add 20ml 20%NaCl in water (w/v) per 100ml 0f 10% ethanol to preserve results for up to 2 days before drying.
Instant Blue stained ClearPAGE 12% SDS 12-
well gel (30 minutes), followed by brief water
wash (5 minutes).
Lane Samples loaded
1-2 E. Coli Extract
3 Bovine Serum Albumin (2μg)
4-8 Broad Range MW Marker
500/200/100/50/25ng per band
9-10 ClearPAGE Two-Color SDS™
Marker 2μl / 4μl
11-12 Chicken Muscle Extract