2-D Electrophoresis

 Sample preparation
buffer composition

 IPG strip rehydration

 1st dimension: IEF run

 IPG strip wash

Equilibration buffer I
Equilibration buffer II

 2nd dimension: SDS-PAGE

 Gel staining (SYPRO ruby)

 Imaging (Fuji imager - 470nm)

Sample preparation
 Consistent protocol

 Limit sample degradation

• fresh cells/tissue
• protease inhibitors
• keep sample cold
• long term storage at –80°C

 Limit sample contamination

• gloves (keratin)
 Sample solubilisation

 Solubilisation/Denaturation buffer

• separate proteins into individual components

• reliable running in the IEF

 Buffer composition
Chaotrope

 Urea (up to 9 M)
 Thiourea (up to 2 M)

• Disrupt of hydrogen and hydrophobic bonds

Note: Urea (if t°>37°C)  cyanate (HN=C=O)

 carbamylation (Lys, Arg)
(R-NH2  R-NH-CO-NH2)
 carbamylation trains
Buffer composition

Reductants

 -mercaptoethanol
 DTT(dithiothreitol)

• Break disulfide bridge

(within or between protein)
Buffer composition
Detergents - surfactants

 SDS (0.1-0.3%)
 CHAPS (up to 4%)

• Disrupt membranes
• Break hydrophobic interactions
• Solubilise lipids
• Release membrane bound proteins

Note: No net charge for IEF (no SDS!)

Soluble in urea
 Buffer composition
Ampholytes (up to 2%)

 Help protein solubilisation

 Scavenge cyanate ions

 Precipitate nucleic acids (during centrifugation)

 Prevent interaction immobilines/protein

 Should represent the pH range desired

 Interfering substances

 Lipids (detergents)

 Proteases (inhibitor cocktails: prokaryote? or eukaryote?)

 Nucleic acids (ultracentrifugation, nucleases)

 Polysaccharides (ultracentrifugation)

 Salts (dialyse;  less than 20 mM)

 Protocol
Bacteria: ~100 g protein
 Protocol
Bacteria: ~100 g protein

TCA (5%) precipitation twice (on ice)

Note: Stay at 4°C (on ice) for these steps!

 Protocol
Bacteria: ~100 g protein

TCA (5%) precipitation twice (on ice)

Wash pellet with ice cold acetone (remove TCA)

Note: Stay at 4°C (on ice) for these steps!

 Protocol
Sample Buffer I
Bacteria: ~100 g protein • 22 mM Tris base
• 28 mM Tris-HCl
TCA (5%) precipitation twice (on ice) • 0.3% SDS
• 200 mM DTT
• Protease inhibitors
Wash pellet with ice cold acetone (remove TCA)
 pH 8.0

Protein pellet resuspended in 40 l Sample Buffer I

Note: Stay at 4°C (on ice) for these steps!

 Protocol
Bacteria: ~100 g protein

TCA (5%) precipitation twice (on Sample

ice) Buffer II
• 24 mM Tris base
Wash pellet with ice cold acetone
• 476 mM(remove
Tris-HCl TCA)
• 50 mM MgCl2
Protein pellet resuspended•in 40 l(2000
1 mg/ml SampleU/ml)Buffer
DNAse II
• 0.25 mg/ml (750 U/ml) RNAse A
 pH 8.0
Add 4 l Sample Buffer II

Note: Stay at 4°C (on ice) for these steps!

 Protocol
Bacteria: ~100 g protein

TCA (5%) precipitation twice (on ice)

Wash pellet with ice cold acetone (remove TCA)

Protein pellet resuspended in 40 l Sample Buffer I

Add 4 l Sample Buffer II

Incubate 10 min on ice

Note: Stay at 4°C (on ice) for these steps!

 Protocol
Loading Buffer

40 mM Tris base
Add 160 l Loading Buffer
8 M urea

4% CHAPS

65 mM DTT

0.01% bromophenol blue

Note: Stay at room t° for these steps!

 Protocol
Rehydration Buffer

8 M urea
Add 160 l Loading Buffer
2% CHAPS
Add 200 l Rehydration Buffer 10 mM DTT

2% ampholytes

0.01% bromophenol blue

Note: Stay at room t° for these steps!

Isoelectric point (pI)
- -

+ +

- -

+ +
 Isoelectric focusing (IEF)
pH gradient

 Ampholytes: (carrier ampholytes)  tube gels

• mixture of amphoteric species with a range of pI values

(small, soluble at pI, minimun interaction with protein, high buffering capacity)

 Immobilines: (~ ampholytes)  IPG strips

• covalently bound to acrylamide gel

• immobilised pH gradient (IPG): gel plastic backed

 IPG strip
Advantages:
• mechanically strong

• pH gradient cannot drift

• load larger amount of sample (dehydrated strip)

Disadvantages:
• membrane/hydrophobic proteins poorly represented on 2D

• some larger proteins lost (size exclusion)

 IPG rehydration

Load 400 l sample per groove (no bubbles!)

Peel off protective film from strip
Place IPG strip gel facing down in groove
Add ~2.5 ml non conductive oil per groove
Rehydrate overnight (~22 hrs) at room temperature
 IEF run
Place wet wicks (H2O) under each end of strip
Set chiller temperature for 20°C (setting ~2.5)
Program power supply
• Number of gels: (1-10)
• Max voltage: 5000 V
• Vhold: 125 V
• Duration: 24 hrs 00 min
• Max current: 80 A/strip
• Volt hours: 80,000 Vh
Anode Cathode
pI
+ -
- - - - -
+ + + + + + +
pH 4 pH 7
 After IEF run

Remove IPG strip from tray

Let oil drip off the strip

Place IPG strip gel facing up in equilibration tray

+ -

Add 10 ml equilibration buffer 1 per tray

 + -

Strip equilibration +

+
-

(in chemical hood) + -

Equilibration Buffer 1 (reduction) (10 ml/strip)

• 6 M urea
• 130 mM DTT (R-S-S-R’  R-SH + R’-SH)
• 30% glycerol
• 1.6% SDS
• 0.002% bromophenol blue
• 45 mM Tris base
• pH 7.0 (acetic acid)
 15 min rocking (room temperature)
 Pour off EB1
 + -

Strip equilibration +

+
-

(in chemical hood) + -

Equilibration Buffer 2 (alkylation) (10 ml/strip)

• 6 M urea
• 135 mM iodoacetamide (R-SH  R-S-CH -CO-NH )
2 2

• 30% glycerol
• 1.6% SDS
• 0.002% bromophenol blue
• 45 mM Tris base
• pH 7.0 (acetic acid)
 15 min rocking (room temperature)
 Pour off EB2
 SDS-PAGE run
• Tris/Tricine/SDS buffer
MW
4 7
-
• IPG strip
+
• MW (gel worm)
• Agarose overlay
• Silicone spacer
• Gasket

10% DuracrylTM gel

(22 cm x 23 cm x 1 mm)
 SDS-PAGE run
Conditions

MW
4
+
7
-
- • Number of gel: (1-10)
• Max voltage: 500 V
• Max power: 1600 mW/gel
• 4°C (setting 10)
• ~19 hrs

200 mM Tris-base
200 mM Tricine
0.4% SDS
Tris/Tricine /SDS
0.45 m filtered!
25 mM Tris-base

10% DuracrylTM gel + pH 8.3 (acetic acid) Tris/acetate

(22 cm x 23 cm x 1 mm)
 Staining
 Coomassie staining
• moderate sensitivity (36-47 ng)
• non specific
• not quantitative

 Silver staining
• sensitive (0.5-1.2 ng)
• time consuming
• non specific
• negative stain some spots

 Fluorescent dye (SYPRO ruby)

• sensitive (1-2 ng)
• specific, quantitative
• end point stain
 Staining protocol

• Fixative (30 min)

(40% Methanol - 10% acetic acid)

• SYPRO ruby (12 hrs)

Protect from light

• Washing (30 min)

(10% Methanol - 6% acetic acid)

• 2% glycerol storage solution

• Store gels at 4°C

 Imaging
• UV detection (300 nm)
• Blue light (470 nm)  5 min
 Fuji Imager
pI
4 5 6 7
kDa

116
97
81
66

55
45
MW

30

21

14
 Image analysis
1 2 3
Samples ran in triplicate

Avg
Build average gel (software)

Differential expression analysis

 IPG strip ranges

IPG strips (3 mm x 18 cm x 0.5 mm)

3 10
 Broad range

4 7
 Medium range 6 11

3.5 4.5 5.5 6.7

 Narrow range
4.0 5.0 6.0
 Broad pH range
(pH 3-10)

pI
3 4 5 6 7 8 9 10
kDa

116
97
81
66

55
45

30

21

14
Medium pH range
(pH 4-7)

pI
4 5 6 7
kDa

116
97
81
66

55
45

30

21

14
 Narrow pH range (1 pH unit)
(4.5-5.5)

(4.0-5.0) (5.0-6.0)
pI
4.0 4.5 5.0 5.5 6.0

116
97
81
66

55
45

MW
(kDa)

30

21

14
 Time line

Sample preparation: 2-3 hrs

IPG strip rehydration: 22 hrs

IEF run: 24 hrs

SDS-PAGE: 19 hrs

Gel staining: 13 hrs

Total: ~ 4 days