Hydration was performed overnight in the Pharmacia reswelling

Two dimensional electrophoresis cassette with 25 ml of a solution containing urea (8 M), CHAPS (2%
w/v), DTE (10 mM), Resolyte pH 3.5-10 (2% v/v) and a trace of
Bromophenol Blue.
ƒIEF: dry strips pH = 4-
4-7
ƒHydratation conditions:urea,
conditions:urea,
thiourea, CHAPS, DTT,
ampholytes, iodoacetamine.
ƒPassive 15h.
ƒ Isoelectrofocussing:
ƒ200v 1h
ƒ500v 1h
ƒ1000v 1h
ƒ5000v 3h

SDS-
SDS-PAGE: 12%
Silver staining

After placing IPG strips, humid electrode wicks, electrodes and

sample cups in position, the strips and cups were covered with low
viscosity paraffin oil. Samples were applied at the cathodic end of the
IPG strips in a slow and continuous manner, without touching the gels.

1
Staining methods Staining methods
Orbital Shaker at 36 rpm
Belly dancer
Horizontal shaker

Staining methods
Organic dye-based methods
Coomassie Blue dyes (R and G types)
Sensitivity low cost
Linear dynamic range ease of use
good compatibility with downstream microchemical characterization
Reproducibility methods

Structure of Coomassie Brilliant Blue G Colloidal Coomassie Blue stains

proteins are gradually stained to an endpoint, without significant
staining of the gel matrix

Limits of Linear range

detection
Coomassie Blue 8–10 ng 10–30-fold
Absorption spectrum of CBB with and without protein
a) protein free dyes
b) 10 μg BSA/ml Colloidal 30–100 ng 10–30-fold
Coomassie Blue

Poor detection sensitivity

Small linear dynamic range

2

Coomassie Blue
Fixing solution: 50% ethanol, 10% acetic acid.
Washing solution: 50% methanol, 5% acetic acid
Stain: Your choice of Coomassie
Destain: 50% methanol, 5% acetic acid.
Storage solution: 5% acetic acid in water.
Coomassie Blue
Fixing solution: 50% ethanol, 10% acetic acid.
Washing solution: 50% methanol, 5% acetic acid
Stain: Coomassie
Destain: 50% methanol, 5% acetic acid.
Storage solution: 5% acetic acid in water.
Fixation
The gel must be fixed by a non-modifying, precipitation procedure such at
the ethanol (or methanol)-acetic acid method used here. If the
protein is not fixed in the gel as a separate step from the staining, the
protein will be washed away and your results will be compromised.
Fixation
The gel must be fixed by a non-modifying, precipitation procedure such at
the ethanol (or methanol)-acetic acid method used here. If the
protein is not fixed in the gel as a separate step from the staining, the
protein will be washed away and your results will be compromised.
Coomassie Blue
Fixing solution: 50% ethanol, 10% acetic acid.
Washing solution: 50% methanol, 5% acetic acid
Stain: Coomassie
Destain: 50% methanol, 5% acetic acid.
Storage solution: 5% acetic acid in water.
Procedure:
1. After electrophoresis, fix the gel by soaking in the fixing
solution overnight. Do not touch the gel with an ungloved hand.
2. Aspirate off the fixing solution and wash the gel twice in the
washing solution, at least 30 min each wash.
3. Aspirate off the washing solution, cover the gel with Coomassie
stain and stain at room temperature for 3 h to overnight.
4. Aspirate off the Coomassie stain.
5. Destain by covering the gel with destain solution. Change the
destain several times until the bands are seen with minimal
background.
6. Store the gel in the storage solution as needed
3
Colloidal Coomassie Blue stains
Fixing Solution: 20% trichloroacetic acid.
Colloidal Coomassie Stain: 0.1% (w/v) Coomassie blue in 20%
(w/v) trichloroacetic acid. Add 0.4 g Coomassie blue G250 to
150 mmL water. Add 150 mL 1M H2SO4 while stirring. Stir
solution 3h to dissolve Coomassie. Filter solution to remove any
insoluble material. Neutralize the sulfuric acid by carefully
adding 33 mL 10 M NaOH while stirring. Allow the solution to
stand 15 min. Add 66 g trichloroacetic acid, stir.
Procedure:
1. After electrophoresis, rinse the gel with water. Remember, do
not touch the gel with an ungloved hand.
2. Aspirate off the water and wash the gel in water for 10 min.
3. Aspirate off the water, cover the gel with 20% (w/v)
trichloroacetic acid fixing solution overnight at room
temperature to fix the proteins.
4. Aspirate off the fixing solution.
5. Cover the gel with the colloidal Coomassie stain and stain for
3 to 4 h with gentle agitation.
6. Aspirate off the Coomassie stain.
7. Wash the acid out of the gel using several changes of water
over ~2 h to enhance contrast.
8. Store the gel in water.

Developing process of film

Black and White films and papers consist of a Silver Halide (AgCl,
AgI, AgBr) layer.These Silver Halides are light sensitive.

Silver Staining -
AgCl Î Ag+ Cl

Silver stain-based methods Silver staining

1. Alkaline methods
The alkaline methods use a diamine complex of silver nitrate in
highly alkaline emviroment.
2. Acidic methods
The acidic methods use silver nitrate in water (weakly acidic
solution) for gel impregnation.

Silver staining is accomplished using silver nitrate in combination

with formaldehyde developer.

Limits of Linear range

detection
Silver staining 0.5 ng 10 fold

4
 Ammoniacal silver staining

• Soaked in ethanol: acetic acid: water (40: 10: 50) for 1 hour.
• Soaked in ethanol: acetic acid: water (5: 5: 90) for 2 hours or overnight.
• Washed in deionized water for 5 min.
• Soaked in a solution containing glutaraldehyde (1%) and sodium acetate (0.5 M) for 30
min.
• Washed 3 times in deionized water for 10 min.
Ammoniacal silver staining • In order to obtain homogeneous dark brown staining of proteins, gels were soaked twice in
a 2,7 naphtalene-disulfonic acid solution (0.05% w/v) for 30 min.
• Rinsed 4 times in deionized water for 15 min.
• Gels were stained in a freshly made ammoniacal silver nitrate solution for 30 minutes.
To prepare 750 ml of this solution, 6 g of silver nitrate were dissolved in 30 ml of
deionized water, which was slowly mixed into a solution containing 160 ml of water, 10
ml of concentrated ammonia (25%) and 1.5 ml of sodium hydroxide (10 N). A transient
brown precipitate might form. After it cleared, water was added to give the final volume.
• After staining, the gels were washed 4 times in deionized water for 4 min.
• The images were developed in a solution containing citric acid (0.01% w/v) and
formaldehyde (0.1% v/v) for 5 to 10 min.
• When a slight background stain appeared, development was stopped with a solution
containing Tris (5% w/v) and acetic acid (2% v/v).

Fixation Ammoniacal silver staining

The gel must be fixed by a non-modifying, precipitation procedure such at
the ethanol (or methanol)-acetic acid method used here. If the
protein is not fixed in the gel as a separate step from the staining, the
protein will be washed away and your results will be compromised.
• Soaked in ethanol: acetic acid: water (40: 10: 50) for 1 hour.
• Soaked in ethanol: acetic acid: water (5: 5: 90) for 2 hours or overnight.
• Washed in deionized water for 5 min.
• Soaked in a solution containing glutaraldehyde (1%) and sodium acetate (0.5 M) for 30
min.
• Washed 3 times in deionized water for 10 min.
• In order to obtain homogeneous dark brown staining of proteins, gels were soaked twice in
a 2,7 naphtalene-disulfonic acid solution (0.05% w/v) for 30 min.
• Rinsed 4 times in deionized water for 15 min.
• Gels were stained in a freshly made ammoniacal silver nitrate solution for 30 minutes.
To prepare 750 ml of this solution, 6 g of silver nitrate were dissolved in 30 ml of
deionized water, which was slowly mixed into a solution containing 160 ml of water, 10
ml of concentrated ammonia (25%) and 1.5 ml of sodium hydroxide (10 N). A transient
brown precipitate might form. After it cleared, water was added to give the final volume.
• After staining, the gels were washed 4 times in deionized water for 4 min.
• The images were developed in a solution containing citric acid (0.01% w/v) and
formaldehyde (0.1% v/v) for 5 to 10 min.
• When a slight background stain appeared, development was stopped with a solution
containing Tris (5% w/v) and acetic acid (2% v/v).

Ammoniacal silver staining

silver nitrate (AgNO3)

Sodium hydroxide (NaOH)
ammonia solution (NH3)

NH3 NH3

+
Ag

5
1. The proteins are exposed to a silver diamine complex in a 2. At such pH, the proteins are anions and can bind the silver
highly alkaline environment (pH = 13). complex by electrostatic interactions with the silver diamine
cationor by amine exchange between one of the ε-amino
group of a lysine.

NH3 NH3

Ag+

Ammoniacal silver staining

• Soaked in ethanol: acetic acid: water (40: 10: 50) for 1 hour.
• Soaked in ethanol: acetic acid: water (5: 5: 90) for 2 hours or overnight.
• Washed in deionized water for 5 min.
• Soaked in a solution containing glutaraldehyde (1%) and sodium acetate (0.5 M) for 30
min.
• Washed 3 times in deionized water for 10 min.
• In order to obtain homogeneous dark brown staining of proteins, gels were soaked twice in
a 2,7 naphtalene-disulfonic acid solution (0.05% w/v) for 30 min.
• Rinsed 4 times in deionized water for 15 min.
• Gels were stained in a freshly made ammoniacal silver nitrate solution for 30 minutes.
To prepare 750 ml of this solution, 6 g of silver nitrate were dissolved in 30 ml of Reduction half-reaction
deionized water, which was slowly mixed into a solution containing 160 ml of water, 10
ml of concentrated ammonia (25%) and 1.5 ml of sodium hydroxide (10 N). A transient
brown precipitate might form. After it cleared, water was added to give the final volume.
• After staining, the gels were washed 4 times in deionized water for 4 min.
• The images were developed in a solution containing citric acid (0.01% w/v) and
formaldehyde (0.1% v/v) for 5 to 10 min. Oxidation half-reaction (formaldehyde)
• When a slight background stain appeared, development was stopped with a solution
containing Tris (5% w/v) and acetic acid (2% v/v).

Ammoniacal silver staining

• Soaked in ethanol: acetic acid: water (40: 10: 50) for 1 hour.
• Soaked in ethanol: acetic acid: water (5: 5: 90) for 2 hours or overnight.
• Washed in deionized water for 5 min.
• Soaked in a solution containing glutaraldehyde (1%) and sodium acetate (0.5 M) for 30
min.
• Washed 3 times in deionized water for 10 min.
• In order to obtain homogeneous dark brown staining of proteins, gels were soaked twice in
a 2,7 naphtalene-disulfonic acid solution (0.05% w/v) for 30 min.
• Rinsed 4 times in deionized water for 15 min.
• Gels were stained in a freshly made ammoniacal silver nitrate solution for 30 minutes.
Oxidation half-reaction (formaldehyde) To prepare 750 ml of this solution, 6 g of silver nitrate were dissolved in 30 ml of
deionized water, which was slowly mixed into a solution containing 160 ml of water, 10
ml of concentrated ammonia (25%) and 1.5 ml of sodium hydroxide (10 N). A transient
brown precipitate might form. After it cleared, water was added to give the final volume.
Reduction half-reaction • After staining, the gels were washed 4 times in deionized water for 4 min.
• The images were developed in a solution containing citric acid (0.01% w/v) and
formaldehyde (0.1% v/v) for 5 to 10 min.
• When a slight background stain appeared, development was stopped with a solution
containing Tris (5% w/v) and acetic acid (2% v/v).

6
 naphtalene-disulfonic acid

glutaraldehyde (1%)
COH COH

COH

Chelate

glutaraldehyde

Ammoniacal silver staining Visible light scanner

• Soaked in ethanol: acetic acid: water (40: 10: 50) for 1 hour.
• Soaked in ethanol: acetic acid: water (5: 5: 90) for 2 hours or overnight.
• Washed in deionized water for 5 min.
• Soaked in a solution containing glutaraldehyde (1%) and sodium acetate (0.5 M) for 30
min.
• Washed 3 times in deionized water for 10 min.
• In order to obtain homogeneous dark brown staining of proteins, gels were soaked twice in
a 2,7 naphtalene-disulfonic acid solution (0.05% w/v) for 30 min.
• Rinsed 4 times in deionized water for 15 min.
• Gels were stained in a freshly made ammoniacal silver nitrate solution for 30 minutes.
To prepare 750 ml of this solution, 6 g of silver nitrate were dissolved in 30 ml of
deionized water, which was slowly mixed into a solution containing 160 ml of water, 10
ml of concentrated ammonia (25%) and 1.5 ml of sodium hydroxide (10 N). A transient
brown precipitate might form. After it cleared, water was added to give the final volume.
• After staining, the gels were washed 4 times in deionized water for 4 min.
• The images were developed in a solution containing citric acid (0.01% w/v) and
formaldehyde (0.1% v/v) for 5 to 10 min.
• When a slight background stain appeared, development was stopped with a solution
containing Tris (5% w/v) and acetic acid (2% v/v).

7
 MS compatible Silver Stain Protocol

MS compatible Silver Stain Protocol

glutaraldehyde (1%)

NH3 NH3

Ag +

Absorption

SYPRO Ruby protein gel stain

Fluorescence SYPRO Ruby protein gel stain is a highly sensitive, ready-to-use

fluorescent stain for proteins in 1-D or 2-D gels. This stain offers many
advantages over silver staining, including a simple staining procedure,
a linear quantitation range over three orders of magnitude and
compatibility with mass spectrometry and microsequencing. Stained
proteins can be viewed with a standard UV or blue-light
transilluminator or with a laser scanner.

Staining gels with SYPRO Ruby protein gel stain is simple: just fix,
stain and wash.

8
 SYPRO Ruby protein gel stain

•High sensitivity
•Fast and easy staining protocols
•Minimal protein-to-protein variation in staining
•Broad linear quantitation range
•Compatibility with subsequent microanalysis
•No nucleic acid staining or polysaccharide staining
•Instrument compatibility

SYPRO Ruby protein gel stain Fluorescence is the result of a three-stage process that
occurs in certain molecules (generally polyaromatic
Dye Ex/Em * Major Features
hydrocarbons or heterocycles) called fluorophores or
Name Applications
fluorescent dyes.

SYPRO 280, 2-D gels, IEF •Highest sensitivity

Ruby 450/610 gels, 1-D (1–2 ng/band;
protein SDS-PAGE comparable to silver
gel stain staining)
•Linear quantitation
range over three
orders of magnitude

9
Luminescence excitation (dashed line) and emission
(solid line) spectra of the SYPRO Ruby protein gel and
blot stains

SyproRuby Silver staining

NADH:ubiquinone reductase precursor
(75, 000-dalton subunit)

SYPRO Ruby protein gel stain shows less protein-to-

protein variation than silver staining.

10
 Metabolic labeling of proteins with [35S]METHIONINE
Intrinsic labeling refers to the incorporation of a
labeled precursor into the protein during synthesis.

Radioactive labeling methods

[35S]METHIONINE Cell culture

1. PULSE-LABELING OF CELLS IN SUSPENSION WITH

[35S]METHIONINE (10 – 30 min)

2. LONG-TERM LABELING OF CELLS WITH [35S]METHIONINE

(16 hr)

Methionine depletion: Methionine labeling:

Resuspend cells at 106 cells/ml in prewarmed pulse-labeling Resuspend cells in a fresh 15-ml centrifuge tube, using 2 ml
medium and incubate 15 min in a 37°C water bath to deplete [35S]methionine working solution.
intracellular pools of methionine.

Methionine-free [35S]methionine
Cell culture medium Cell culture

Phosphate depletion:

Preincubate Cells at 106 cells/ml for 30 min in serum-free and

phosphate-free medium to deplete intracellular pools of
ATP.

Radioactive labeling methods

[32P] Orthophosphate Phosphate-free
Cell culture medium
Pi

11
Phosphate labeling: Cassette and autoradiography
Autoradiography uses X-ray film to visualize and quantitate
Subsequently, cells are incubated for 3 h in medium containing [32 radioactive molecules
orthophosphate.

[32P] Orthophosphate
Cell culture
Pi Pi

Double-coated films: Single-coated films:

high-energy β particles emitted by 32P and 125I Autoradiography films that are single-emulsion coated are
Films are normally used with calcium tungstate (CaWO4) optimized for use with medium-energy radioisotopes—e.g.,
14C, 35S, and 33P.
intensifying screens at reduced temperature (-70°C).

blue light emitted from the intensifying screens

The Chemical Process in Black and White Photography Film developing

Black and White films and papers consist of a Silver Halide (AgCl,
AgI, AgBr) layer.These Silver Halides are light sensitive.

• Exposure A halo image is formed on the negative/photo paper

inside the silver halide emulsion layer.

• Developing All the silver halides that were not exposed to light
are extracted from the negative/photo paper leaving the
exposed parts as black metal silver

• Stop The stop solution stops the developing process.

• Fix The fixing solution fixes the developed silver onto the
negative/photo paper.

• Washing The extracted silver halides are washed away from the
negative/photo paper with water.

12
Developing process of film Fluorescence: short-lived luminescence, 10-8 ~ 10-4 s
Black and White films and papers consist of a Silver Halide (AgCl,
Phosphorescence: long-lived luminescence, 10-4 ~ 102
AgI, AgBr) layer.These Silver Halides are light sensitive.

AgCl Î Ag+ Cl
-

Fluorescence Scanner: Typhoon Fluorescence detection

13
Confocal Summary of protein expression profile analysis

Susana Cristobal
Bioinformatik, 4p. KTH

DIGE

Difference gel electrophoresis (DIGE)

Susana Cristobal
Bioinformatik, 4p. KTH

14
 The chemistry of labelling proteins with CyDye DIGE
Fluor minimal dyes
The ratio used ensures that the dyes label approximately 1–2% o
lysine residues so each labelled protein carries only one dye labe
and is visualised as a single protein spot.
giving equivalent levels of sequence coverage
compared to direct identification from unlabelled
proteins
CyDye DIGE Fluor minimal dyes have an NHS ester reactive grou
and are designed to form a covalent bond with the epsilon amino g
of lysine in proteins via an amide linkage.
Each CyDye DIGE Fluor minimal dye, when coupled to a protein,
add approximately 500 Da to the mass of the protein. This mass s
does not effect the pattern visible on a 2-D gel.
• Ensure the sample is prepared in a buffer that is compatible with
the labelling method.
• Ensure the sample protein concentration is 5-10 mg/ml.
• Ensure the sample pH lies in the range pH 8.0–9.0.
• Create a pooled internal standard from all samples for inclusio
every gel.
• The CyDye DIGE Fluor minimal dyes should be reconstituted to
form a stock solution.
• For labelling, an aliquot of the CyDye DIGE Fluor minimal dye
stock solution should be diluted to a concentration of 400 ρmol/µ
• The ratio of protein to CyDye DIGE Fluor minimal dye should be
maintained at 50 µg: 400 ρmol.
• New protein samples should be checked for successful labelling
The lysine amino acid in proteins carries an intrinsic +1 charge at
neutral or acidic pH. CyDye DIGE Fluor minimal dyes also carry a
+1 charge which, when coupled to the lysine, replaces the lysine’s
+1 charge with its own, ensuring that the pI of the protein does n
significantly alter.
Spot picking: the total protein should be visualized using a
Poststaining method and the position of spots for picking bas
on this new image.
SYPRO™ Ruby
Coomassie™
silver staining
15
 An analytical gel can be post-stained and used directly for spot p
The SYPRO Ruby and CyDye DIGE Fluor minimal dye images fro
this gel are matched, locating spots for picking on the SYPRO Rub
image. More commonly, a separate preparative gel is generated,
a high loading of unlabelled protein. This gel is post-stained and th
matched back to the analytical set of gels. This allows the spots se
for picking to be linked between the analytical data and the poststa
gel image.

Why pooled internal standard ? Why pooled internal standard ?

1 System variation: gel-to-gel variation can result from differe
in electrophoretic conditions
2 Inherent biological variation:
Inherent biological variation arises from intrin
differences that occur within populations.

induced biological changes

Why pooled internal standard ? Why pooled internal standard ?

Intra-gel co-detection - All samples are co-detected with the in
standard.

B1
A1

B0 B0
A0 A0

A0+B0+A1+B1+A2+B2 A0+B0+A1+B1+A2+B2
B2
A2

16
Inter-gel matching - only the internal standards need to be mat
These are derived from the same sample and therefore this aid
matching.

B0
A0
A0+B0+A1+B1+A2+B2

17