2D_Electrophoresis_11-04-04

8/8/2019 2D_Electrophoresis_11-04-04

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Sample preparation

buffer composition

IPG strip rehydration

1 st dimension: IEF run

IPG strip washEquilibration buffer IEquilibration buffer II

2 nd dimension: SDS-PAGE

Gel staining (SYPRO ruby)

Imaging (Fuji imager - 470nm)

2-D Electrophoresis


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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)


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Sample solubilisation

Solubilisation/Denaturation buffer

separate proteins into individual components

reliable running in the IEF


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Buffer composition

Chaotrope

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

Disrupt of hydrogen and hydrophobic bonds

Note: Urea (if t>37C) cyanate (HN=C=O)carbamylation (Lys, Arg)

(R-NH 2 R-NH-CO-NH 2)

carbamylation trains


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Reductants

-mercaptoethanolDTT(dithiothreitol)

Break disulfide bridge(within or between protein)

Buffer composition


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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


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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

Buffer composition


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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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Bacteria: ~100 g protein

TCA (5%) precipitation twice (on ice)

Protocol

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


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Wash pellet with ice cold acetone (remove TCA)

Protocol



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Wash pellet with ice cold acetone (remove TCA)

Protein pellet resuspended in 40 l Sample Buffer I

Protocol


22 mM Tris base 28 mM Tris-HCl 0.3% SDS 200 mM DTT Protease inhibitors pH 8.0

Sample Buffer I


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Wash pellet with ice cold acetone (remove TCA)Protein pellet resuspended in 40 l Sample Buffer I

Add 4 l Sample Buffer II

Protocol


24 mM Tris base

476 mM Tris-HCl 50 mM MgCl 2 1 mg/ml (2000 U/ml) DNAse I 0.25 mg/ml (750 U/ml) RNAse A

pH 8.0

Sample Buffer II


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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

Protocol



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Add 160 l Loading Buffer

Protocol

40 mM Tris base

8 M urea

4% CHAPS

65 mM DTT

0.01% bromophenol blue

Loading Buffer

Note: Stay at room t for these steps!


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Add 160 l Loading Buffer

Add 200 l Rehydration Buffer

Protocol

8 M urea

2% CHAPS

10 mM DTT

2% ampholytes

0.01% bromophenol blue

Rehydration Buffer

Note: Stay at room t for these steps!


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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

Isoelectric focusing (IEF)

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


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IPG stripAdvantages:

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 l sample per groove (no bubbles!)

Place IPG strip gel facing down in groove

Add ~2.5 ml non conductive oil per groove

Peel off protective film from strip

Rehydrate overnight (~22 hrs) at room temperature


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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

pI

+

+ --- - - -

+ + + + + ++-

pH 4 pH 7

CathodeAnode

IEF run

Place wet wicks ( H2O) under each end of stripSet chiller temperature for 20C (setting ~2.5)


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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


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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)

Strip equilibration

15 min rocking (room temperature)

+ -

+ -

+ -

+ -

(in chemical hood)

Pour off EB1


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Strip equilibration

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-CH 2-CO-NH 2)

+ -

+ -

+ -

+ -

(in chemical hood)

15 min rocking (room temperature)

Pour off EB2


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SDS-PAGE run

MW4 7+ -

10% Duracryl TM gel(22 cm x 23 cm x 1 mm)

Tris/Tricine/SDS buffer IPG strip MW (gel worm) Agarose overlay Silicone spacer Gasket


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StainingCoomassie 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

Fixative (30 min)

SYPRO ruby (12 hrs)

Washing (30 min)

2% glycerol storage solution

Store gels at 4C

(40% Methanol - 10% acetic acid)

(10% Methanol - 6% acetic acid)

Protectfrom

light


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Imaging UV detection (300 nm)

Blue light (470 nm) 5 min

Fuji Imager

4 7pI

5 6kDa

116978166

55

45

30

21

14

MW


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Image analysis

Samples ran in triplicate

Build average gel (software)

Differential expression analysis

1 2 3

Avg


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IPG strip ranges

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

Narrow range

Medium range

Broad range

4 7

3.5 4.5 5.5 6.7

4.0 5.0 6.0

3 10

611


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Broad pH range(pH 3-10)

1169781

6655

45

30

21

14

kDa

pI3 1074 5 6 8 9


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Medium pH range(pH 4-7)

1169781

66

55

45

30

21

14

kDa

pI4 75 6


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Narrow pH range (1 pH unit)

5.5 6.05.04.54.0

116

66

97

55

81

30

45

21

14

pI

MW(kDa)

(4.5-5.5)

(4.0-5.0) (5.0-6.0)


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Time line

Sample preparation:

IPG strip rehydration:

IEF run:

SDS-PAGE:

Gel staining:

Total: ~ 4 days

2-3 hrs

22 hrs

24 hrs

19 hrs

13 hrs

Documents

2D_Electrophoresis_11-04-04