2D Electrophoresis

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2-D Electrophoresis
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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)
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Sample preparation

• Consistent protocol
• Limit sample degradation
• fresh cells/tissue
• protease inhibitors
• keep sample cold
• long term storage at 80C
• Limit sample contamination
• gloves (keratin)

3
Sample solubilisation

• Solubilisation/Denaturation buffer
• separate proteins into individual components
• reliable running in the IEF

4
Buffer composition

• Chaotrope
• Urea (up to 9 M)
• Thiourea (up to 2 M)
• Disrupt of hydrogen and hydrophobic bonds
• Note Urea (if tgt37C) ? cyanate (HNCO)
• ? carbamylation (Lys, Arg)
• (R-NH2 ? R-NH-CO-NH2)
• ? carbamylation trains

5
Buffer composition

• Reductants
• b-mercaptoethanol
• DTT(dithiothreitol)
• Break disulfide bridge
• (within or between protein)

6
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

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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

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Interfering substances

• Lipids (detergents)
• Proteases (inhibitor cocktails prokaryote? or
  eukaryote?)
• Nucleic acids (ultracentrifugation, nucleases)
• Polysaccharides (ultracentrifugation)
• Salts (dialyse ? less than 20 mM)

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Protocol
Bacteria 100 mg protein
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Protocol
Bacteria 100 mg protein TCA (5) precipitation
twice (on ice)
Note Stay at 4C (on ice) for these steps!
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Protocol
Bacteria 100 mg protein TCA (5) precipitation
twice (on ice) Wash pellet with ice cold acetone
(remove TCA)
Note Stay at 4C (on ice) for these steps!
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Protocol
Bacteria 100 mg protein TCA (5) precipitation
twice (on ice) Wash pellet with ice cold acetone
(remove TCA) Protein pellet resuspended in 40 ml
Sample Buffer I
Note Stay at 4C (on ice) for these steps!
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Protocol
Bacteria 100 mg protein TCA (5) precipitation
twice (on ice) Wash pellet with ice cold acetone
(remove TCA) Protein pellet resuspended in 40 ml
Sample Buffer I Add 4 ml Sample Buffer II
Note Stay at 4C (on ice) for these steps!
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Protocol
Bacteria 100 mg protein TCA (5) precipitation
twice (on ice) Wash pellet with ice cold acetone
(remove TCA) Protein pellet resuspended in 40 ml
Sample Buffer I Add 4 ml Sample Buffer
II Incubate 10 min on ice
Note Stay at 4C (on ice) for these steps!
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Protocol
Add 160 ml Loading Buffer
Note Stay at room t for these steps!
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Protocol
Add 160 ml Loading Buffer Add 200 ml Rehydration
Buffer
Note Stay at room t for these steps!
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Isoelectric point (pI)
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Isoelectric focusing (IEF)

• pH gradient
• Ampholytes (carrier ampholytes) ? tube gels
• mixture of amphoteric species with a range of pI
  values
• Immobilines ( ampholytes) ? IPG strips
• covalently bound to acrylamide gel
• immobilised pH gradient (IPG) gel plastic backed

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

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IPG rehydration
Load 400 ml 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
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IEF run
Place wet wicks (DH2O) under each end of strip
Set chiller temperature for 20C (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 mA/strip
• Volt hours 80,000 Vh

Anode
pI
Cathode

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pH 4
pH 7
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After IEF run
Let oil drip off the strip
Add 10 ml equilibration buffer 1 per tray
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Strip equilibration
(in chemical hood)

• Equilibration Buffer 1 (reduction) (10 ml/strip)
• 6 M urea
• 130 mM DTT
• 30 glycerol
• 1.6 SDS
• 0.002 bromophenol blue
• 45 mM Tris base
• pH 7.0 (acetic acid)

(R-S-S-R ? R-SH R-SH)

• 15 min rocking (room temperature)

• Pour off EB1

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Strip equilibration
(in chemical hood)

• Equilibration Buffer 2 (alkylation) (10 ml/strip)
• 6 M urea
• 135 mM iodoacetamide
• 30 glycerol
• 1.6 SDS
• 0.002 bromophenol blue
• 45 mM Tris base
• pH 7.0 (acetic acid)

(R-SH ? R-S-CH2-CO-NH2)

• 15 min rocking (room temperature)

• Pour off EB2

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SDS-PAGE run
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• Tris/Tricine/SDS buffer

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• IPG strip

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• MW (gel worm)

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• Agarose overlay

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• Silicone spacer

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• Gasket

10 DuracrylTM gel (22 cm x 23 cm x 1 mm)
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SDS-PAGE run

• Conditions
• Number of gel (1-10)
• Max voltage 500 V
• Max power 1600 mW/gel
• 4C (setting 10)
• 19 hrs

10 DuracrylTM gel (22 cm x 23 cm x 1 mm)
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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

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Staining protocol
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Imaging

• UV detection (300 nm)
• Blue light (470 nm) ? 5 min
• ? Fuji Imager

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Image analysis
Samples ran in triplicate Build average gel
(software) Differential expression analysis
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IPG strip ranges
IPG strips (3 mm x 18 cm x 0.5 mm)

• Broad range

• Medium range

• Narrow range

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Broad pH range(pH 3-10)
pI
3
10
7
4
5
6
8
9
kDa
116
97
81
66
55
45
30
21
14
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Medium pH range(pH 4-7)
pI
4
7
5
6
kDa
116
97
81
66
55
45
30
21
14
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Narrow pH range (1 pH unit)
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Time line
Sample preparation IPG strip rehydration IEF
run SDS-PAGE Gel staining
2-3 hrs 22 hrs 24 hrs 19 hrs 13 hrs
Total 4 days