TITLE: PROTEIN GEL

STAINING TECHNIQUES
Presented By:
Shajila Faiz
(M-Phill-1st Semester)

Content

Protein Staining Technique

Principal of Protein Gel Staining
Types of Total Protein Gel Stain
Coomassie Blue Stains
Silver Stains
Fluorescent Stains

Other than Total Protein Gel Stain

Functional Group Specific Stains
Zinc Stains

Troubleshooting
References
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PROTEIN GEL STAINING

TECHNIQUE

Proteins separated by gel electrophoresis can be

visualized using different staining procedures. The
choice of staining technique depends on the
availability of imaging equipment in the lab in
many cases.

There are different staining methods of in gel

detection each with its specific advantages and
disadvantages
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Principle:
AA protein
protein staining
staining technique
technique should
should offer
offer the
the following:
following:
1.
1. High
High sensitivity
sensitivity and
and reproducibility.
reproducibility.
2.
2. Wide
Wide linear
linear dynamic
dynamic range.
range.
3.
3. Compatibility
Compatibility with
with downstream
downstream technologies
technologies such
such as
as
protein
protein extraction
extraction and
and assay,
assay, blotting,
blotting, or
or mass
mass
spectrometry.
spectrometry.
4.
4. Robust,
Robust, fast,
fast, and
and uncomplicated
uncomplicated protocol.
protocol.
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Basic Procedure

Staining procedure usually involve the

following three steps:

1. Protein fixation, usually in acidic

methanol, ethanol or alcohol.

2. Staining, addition of dye in proteins.

3. Destaining, washing to remove excess

dye.
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Total Protein Stain

Total
Totalprotein
proteinstains
stainsallow
allow
visualization
of
the
visualization of the
protein
proteinpattern
patternin
inthe
the
gel.It
is
done
by
the
gel.It is done by the
stains/dyes
stains/dyeswhich
whichare
are
listed
below:
listed below:

Coomassie
CoomassieStains
Stains

Silver
SilverStains
Stains

Fluorescent
FluorescentStains
Stains

A.Coomaissie Stains
General
General Procedure:
Procedure:
1.Washing:water
1.Washing:water wash
wash step
step is
is necessary
necessary to
to remove
remove
remains
remains of
of SDS,
SDS, which
which interferes
interferes with
with dye
dye binding.
binding.
.
22..Staining:The
.Staining:The addition
addition of
of staining
staining reagent
reagent about
about 11 hour
hour

3:Destaining
3:Destaining aa water
water or
or simple
simple methanol:
methanol: acetic
acetic acid
acid
destaining
destaining step
step is
is used
used to
to wash
wash away
away excess
excess unbound
unbound dye
dye
from
from the
the gel
gel matrix.
matrix.
Protein
Protein can
can be
be analyzed
analyzed by
by mass
mass spectrometry.
spectrometry.
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Types
Types of
of Coomassie
Coomassie Stains
Stains
There
There are
are two
two types
types of
of Coomassie
Coomassie Brilliant
Brilliant Blue:
Blue:
1.R-250
1.R-250 (R
(R for
for reddish):
reddish):
Which
Which offers
offers shorter
shorter staining
staining times.
times.
2.G-250
2.G-250 (G
(G for
for greenish):
greenish):
Which
Which is
is available
available in
in more
more sensitive
sensitive and
and
environmentally
environmentally friendly
friendly formulations.
formulations.
The color of both dyes depends on acidity of the
The color of both dyes depends on acidity of the
solution
solution
At a pH of less than 0 the dye has a red color
At a pH of less than 0 the dye has a red color
At a pH around 1 the dye has a green color
At a pH around 1 the dye has a green color
At a pH of above 2 the dye has a bright blue color
At a pH of above 2 the dye has a bright blue color
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Advantages:
Coomassie
Coomassie dyes,
dyes, provides
provides aa happy
happy medium
medium for
for protein
protein
staining,
staining, having
having relatively
relatively high
high sensitivity
sensitivity and
and being
being
simple
simple to
to perform,
perform,
They
They are
are the
the favorite
favorite stains
stains for
for mass
mass spectrometry
spectrometry and
and
protein
protein identification.
identification.
Coomassie
Coomassie Blue
Blue G-250
G-250 is
is aa nonhazardous
nonhazardous formulation
formulation
that
that requires
requires only
only water
water for
for rinsing
rinsing and
and destaining.
destaining.
As
As the
the colloidal
colloidal dyes
dyes does
does not
not penetrate
penetrate the
the gel,
gel, so
so no
no
destaining
destaining is
is required,
required, resulting
resulting in
in higher
higher reproducibility.
reproducibility.

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Disadvantages

This
This technique
technique has
has aa detection
detection limit
limit of
of only
only about
about 100
100 ng
ng ,,
making
making visualization
visualization of
of low
low abundance
abundance proteins
proteins difficult.
difficult.

This
This technique
technique also
also has
has aa low
low reproducibility
reproducibility and
and is
is due
due to
to the
the
difficulty
difficulty in
in standardizing
standardizing the
the destaining
destaining step.
step.

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The two Coomassie blue dyes,R-250andG-250, differ by

only two methyl groups. In function, the more popular
Coomassie R-250is more sensitive whileG-250is more
convenient.

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Gel staining with Coomassie dye.Two-fold dilutions of protein extracts

were run on an Invitrogen NativePAGE 312% Bis-Tris Protein Gel
using a Mini Gel Tank. Following electrophoresis, the gel was stained
with Coomassie dye and imaged using a flatbed scanner. Lanes 1 and
10: blank; lanes 2 and 6: 5 L Invitrogen NativeMark Unstained
Protein Standard; lanes 3, 4 and 5: 10, 5, and 2.5 g spinach
chloroplast extract; lanes 7, 8, and 9: 10, 5, and 2.5 g bovine
mitochondrial extract.
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 1.Metallic silver deposited on the surface

of a gel over the protein bands.
2.Silver ions interact and bind with
certain protein functional groups.
3.Various sensitizer and enhancer are
essential for(controlling efficiency of silver
ion binding to proteins effective
conversion of the bound silver to metallic
silver)
4.Silver ions are reduced to metallic
silver, resulting in a brown-black color.

Chemistr
Chemistr
y:
y:

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Gel staining with silver stain. Samples were separated on an Invitrogen

NuPAGE 412% Bis-Tris Protein Gel and stained with the Invitrogen
SilverXpress Kit. Lanes 15: Invitrogen Mark12 Unstained Standard
(blend of 12 purified

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Silver
Silver staining
staining is
is the
the most
most sensitive
sensitive
colorimetric
colorimetric method
method for
for detecting
detecting total
total
protein
protein
Ability
Ability to
to detect
detect II ng
ng of
of protein
protein
Sensitivity
Sensitivity of
of Silver
Silver stains
stains are
are more
more than
than
Coomassie
Coomassie and
and is
is equivalent
equivalent to
to most
most
fluorescent
fluorescent stains.
stains.
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ItIt is
is relatively
relatively expensive
expensive due
due to
to its
its reagents
reagents
and
and wastage.
wastage.

ItIt has
has aa high
high background
background
This
This technique
technique is
is time-consuming,
time-consuming, complex,
complex,
and
and do
do not
not offer
offer sufficient
sufficient reproducibility
reproducibility for
for
quantitative
quantitative analysis.
analysis.
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Simple Dye Binding

Technique:

Most fluorescent stains

involve simple dye-binding
mechanisms instead of
chemical reactions that
alter the protein functional
groups.
These stains fulfill almost
all the requirements for an
ideal protein stain.

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1.Fluorescent
Dyes are highly
sensitive.

2.They are
compatible with
mass
spectrometry.

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In comparison to Coomassie or silver staining techniques,

fluorescent dyes are more expensive because they require
either a CCD (charge-coupled device) camera or fluorescence
scanner for gel imaging.

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Protein gel stained with fluorescent dyes.2D gel stained with

Invitrogen SYPRO Ruby protein gel stain and Invitrogen
Pro-Q Emerald 300 reagent. Cohn fractions II and III from cow
plasma were combined and resolved on a 2D gel. The gel was
first stained with Pro-Q Emerald 300 reagent (left), followed by
staining with the SYPRO Ruby stain (right).

21

Zinc stains
dye
polyacrylamid
e gel in which
there are no
proteins.
22

Chemistry:

Zinc ions form complex with imidazole which precipitates in the

gel matrix except where SDS-saturated proteins are located.

The milky-white precipitate causes the background opaque while

the protein bands remain clear. The process is short (about 15
minutes), and the gel can be photographed by viewing it over a
dark background.
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It detects less
than 1 ng of
protein

There is no
fixation steps
The stain is
easily removed,
making this
method
compatible with
western blotting.

24

Gel staining with zinc stain. A 2-fold dilution series of a protein mixture was separated
by protein gel electrophoresis using a 15-well mini gel. Subsequently the gel was
stained using the Thermo Scientific Pierce Zinc Reversible Stain Kit, and then
photographed with the gel placed over a dark blue background. The sensitivity on this
gel is 0.25 ng, as indicated by the bands that are visible in the last lan

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Advantages

Greater Sensitivity than silver stains.

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Functional groupspecific stains

Sometimes it is desirable to detect a subset of
proteins rather than all of the proteins in a
sample.

Example:
Glycoproteins and phosphoproteins (i.e.,
polysaccharides and phosphate groups,
respectively). When a dye-binding or colorproducing chemistry can be designed to
detect one of these functional groups, it can
be used as the basis for a specific gel stain
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Chemistry

This involves
1. Activation of the carbohydrate into a
reactive group.
2. This can be done by fixing the proteins in
the gel
3.Oxidizing the sugar residues with sodium
meta-periodate. The resulting aldehyde
groups can then be reacted with an aminecontaining dye.
This method is known as the periodate acid
Schiff (PAS) technique.
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Phosphoprotein and total protein visualization in a 2D gel. Protein lysates obtained from
a Jurkat T-cell lymphoma line were separated by 2D gel electrophoresis and
subsequently stained with Invitrogen Pro-Q Diamond phosphoprotein gel stain (blue)
followed by SYPRO Ruby protein gel stain (red). The gel was dried and imaged on an
FLA-3000 scanner (Fuji). Shown is a digitally pseudocolored composite overlaid image.

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Troubleshooting
Problem

Cause

Solution

Bands not visible

No protein in gel

Stain with another method to confirm there is protein

Imaging system malfunctioning

Check instrument manual for troubleshooting, or

contact imaging instrument manufacturer

Incorrect imaging parameters were used

Check instrument manual

Dirty staining trays

Clean staining trays and other equipment with

laboratory glassware cleaner

Insufficient stain volume

Follow recommendations for stain volume (appropriate

to gel size)

Insufficient staining time

Increase staining time

Reuse of staining solution

Repeat staining protocol with fresh staining solution

Dirty equipment or staining trays

Clean staining trays and other staining equipment

with laboratory glassware cleaner

Too much time in staining solution

Restrict duration of incubation in staining solutions as

recommended in protocol

Poor staining sensitivity

High or uneven background

staining

Wash gel in water or respective destaining solution for

30 min

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References

Diezel W, Kopperschlger G, Hofmann E. An improved procedure for

protein staining in polyacrylamide gels with a new type of
Coomassie Brilliant Blue.Anal. Biochem.48(2), 617620 (1972).
O'Farrell PH. High resolution 2D electrophoresis of proteins.J. Biol.
Chem.250(10), 40074021 (1975).
Chevalier F, Rofidal V, Vanova P, Bergoin A, Rossignol M. Proteomic
capacity of recent fluorescent dyes for protein
staining.Phytochemistry65(11), 14991506 (2004).
Rabilloud T. A comparison between low background silver diammine
and silver nitrate protein stains.Electrophoresis13(7), 429439
(1992).
Fernandez-Patron C, Castellanos-Serra L, Hardy Eet
al.Understanding the mechanism of the zinc-ion stains of
biomacromolecules in electrophoresis gels: generalization of the
reverse-staining technique.Electrophoresis19(14), 23982406
(1998).

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