Bio-Rad Total Protein Blot Stains - Manual (Page 5)

25% ethanol, 12.5% trichloroacetic acid
10% ethanol, 7% acetic acid
50% ethanol, 3% acetic acid
40% ethanol, 10% acetic acid
For IEF gels, fix gel in 40% methanol, 10% trichloroacetic acid
for 3 hours, then wash gel in water 3 times for 10 min each.
(No fixation is required for 1-D gels).
b) Remove the wash solution and cover the gel with SYPRO Ruby
protein gel stain. The volumes used for typical gels are given
below. In general use ~ 10 times the volume of the gel. Using too
little stain will reduce sensitivity.
Gel Size Volume of Stain
8 x 10 cm 50 ml
16 x 20 cm 330 ml
20 x 20 cm 500 ml
c) Stain the gel with continuous gentle agitation for at least 3 hrs for
maximal sensitivity. Specific staining can be seen in 30–90 min.
For convenience, gels may be left in the stain solution overnight
(16–18 hrs) without overstaining.
d) Rinse the gel in 10% methanol (or ethanol), 7% acetic acid for
30–60 min. This rinse step decreases background fluorescence.
e) Wash gel in water before imaging.
2.2 Viewing and Imaging a 1D or 2D Gel
SYPRO Ruby protein gel stain has two excitation peaks at ~ 280 nm
and ~ 450 nm and has an emission maxima near 610 nm. Stained pro-
teins can be visualized using variety of excitation sources including a
300 nm UV or blue-light transilluminator, or laser-based systems.
SYPRO Ruby protein gel stain also has exceptional photostability and a
long emission lifetime, allowing for long exposure times while minimiz-
ing background fluorescence. For the superior resolution required for
2D gel analysis, the Bio-Rad Molecular Imager™ FX or Fluor-S™
MultiImagers are recommended.
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PART 2: SYPRO Ruby protein gel stain
Section 1
General Information
1.1 Introduction
SYPRO Ruby protein gel stain is a ready–to–use, sensitive,
f l u o r e s cent stain for detecting proteins separated by polyacry-
lamide gel electrophoresis (PAGE). While specially formulated for
analysis of proteins in 2-D polyacrylamide gels, SYPRO Ruby pro-
tein gel stain also stains 1D gels of different buffer compositions
including Tris-Glycine-SDS, Tris-Tricine and non-denaturing PAGE as
well as IEF gels. The stain does not interfere with subsequent analysis of
proteins by Edman-based sequencing or mass spectrometry and is quanti-
tative over three orders of magnitude. Use SYPRO Ruby protein gel to
stain difficult to stain proteins such as glycoproteins and lipoproteins.
This stain will not stain nucleic acids.
1.2 Product Description
SYPRO Ruby protein gel stain is a 1x pre-mixed solution, and
comes in three package sizes with instructions. The 200 ml size will stain
~ 4 mini gels, the 1 L size will stain ~20 mini gels or 2–3 large gels, the
5 L size will stain ~ 100 minigels or 10–15 large gels.
170-3125 SYPRO Ruby protein gel stain, 1 L
170-3126 SYPRO Ruby protein gel stain, 200 ml
170-3138 SYPRO Ruby gel stain, 5 L
Section 2
Instructions
2.1 Staining
a) Remove the gel from the gel cassette or plates. Place it in a clean
plastic container (see Section1.4). For 2-D gels, wash for 30 min
in one of the following gel fixing solutions:
10% methanol, 7% acetic acid
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SYPRO Ruby protein gel stain SYPRO Ruby protein gel stain
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