Two-Dimensional Gel Electrophoresis (2-DGE)

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Two

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Dimensional Gel Electrophoresis (2

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DGE)

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Introduction

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Sample preparation

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First dimension: Isoelectric focusing

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Second dimension: SDS

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PAGE

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Detection of protein spots: staining

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Imaging analysis & 2D Gel databases

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Two

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Introduction

* The goal of two-dimensional electrophoresis is to separate and display all gene products present.

* It is the only method currently available which is capable of simultaneously separating thousands of proteins.

* The first dimension of 2-DE - isoelectric focusing (IEF). - pH gradient support

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* The second dimension of 2-DE - sodium dodecyl sulfate PAGE (SDS-PAGE).

- SDS as surfactant. - Molecular mass.

* High resolution from independent protein parameters.

* In the early 1970s, first use of 2-DE to separate serum proteins.

* Drawbacks

- Poor reproducibility - Limited sample loading * Progress

- Chemical or Mass spectrometric analysis

- Immobilized pH gradient (IPG) gels - More sensitive detection procedures - Computer software

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Areas needed major progress

* Sample preparation and solubilization

- Insoluble sample: membrane and nuclear proteins

- Proteins from highly resistant tissues like hair and skin * Low abundance proteins

* Prefractionation * High sample loads * Basic proteins * Quantitation

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Sample preparation

* A critical step in 2-DE.

* Solubilization, denaturation, reduction & removal of non-protein sample components.

* Standard sample solubilization solution:

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Sample preparation

* Optional modifications for more proteins displayed, shorter focusing time, and more sharply focused spots:

- Chaotropes: 8M Urea

2M Thiourea & 7M Urea - Surfactants: 4% CHAPS

2% CHAPS & 2% Sulfobetaine 3-10 - Reducing agents: 100mM DTT

2mM TBP (Tributyl phosphine) and/or 65mM DTT

NCH₂CH₂CH₂SO₃ CH₂ CH₃ (CH₂)₃ R CHAPS Sulfobetaine HS C C SH H OH H OH DTT H₂N NH₂ O H₂N NH₂ S Urea Thiourea

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Sample preparation

Sequential extraction of proteins:

- decreasing spot numbers, yet still display all the proteins.

SAMPLE

Insoluble pellet 1

Insoluble pellet 2

40 mM Tris

Supernatant 1

8M urea, 4% CHAPS, 2mM TBP,

0.2% ampholytes, 40 mM Tris

Supernatant 2

5M urea, 2M thiourea, 2% CHAPS,

2% SB 3-10, 2mM TBP, 0.2% ampholytes, 40 mM Tris

Supernatant 3

hydrophilic highly hydrophobic hydrophobic

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Sample preparation

* Protein of medium abundance, occurring in about 1,000 copies per cell, or less than two picomoles (100 ng) in one liter of cell culture.

* Preparation method: - Differential extraction - Removal of nucleic acids - Subcellular fractionation - Chromatography

- Immunoprecipitation - Affinity-based selection - Others

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Sample loading on IPG gels

* Choice of first-dimension IPG strips

BIO-RAD PROTEAN IEF cell

Amersham Pharmacia Biotech

IPGphor

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Sample loading on IPG gels

* Protein loadings for gels (guide only)

* The narrower the pH range of IPG, the more protein should be loaded.

* For single pH unit IPG’s, this is can be as much as 4-5x more.

1-3 mg total protein Preparative load (comassie) large gel

200-500 µg total protein Preparative load (comassie) mini gel

100-200 µg total protein Analytical load (silver or sypro) large gel

10-50 µg total protein Analytical load (silver or sypro) mini gel

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Sample loading on IPG gels

* IPG are supplied dry and needed to be rehydrated to its original thickness (0.5 mm) prior to IEF.

- Cup loading of sample after IPG rehydration (current & absorption) - Passive in-gel rehydration of sample (absorption)

- Active in-gel rehydration of sample (current & absorption) * Cup loading method recommended for sample:

- containing high level of DNA/RNA or other large molecules such as cellulose.

- analytical serum samples - basic IPG’s eg 7-10

- high in glycoprotein - purified proteins

* Choose available pH range and size IPG (7cm, 11 cm, 18cm, & 24 cm) * Storage at –80ºC.

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SDS

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PAGE gels

* IPG equilibration & casting IPG on the second dimension.

Final volume 100 ml 10 ml (2.5%) Alkylation (prevents bonds rejoining, compatibility for MS) 25% Acrylamide soln. 2.5 ml (5mM) Reduction (breaks the

disulfide bonds) 200 mM TBP

40 ml (20%)

Inhibits electroendosmosis in the viscosity stops water

transfer across the IPG

50% glycerol

20 ml (1x) Buffering (pH 8.8)

5x Tris/HCl gel buffer

2 g (2%) Solubilization of proteins SDS 36g urea (6M) Denaturation and solubilization of proteins Urea AMOUNT/FINAL CONC. FUNCTION REAGENTS

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SDS

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PAGE gels

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Staining

* Sensitive, quantitative and MS-compatible.

- Comassie brilliant blue R250 staining (30-100 ng) - Colloidal comassie blue G250 staining (30-100 ng) - Diamine silver stain (1-10 ng) : gluteraldehyde - Silver nitrate stain (1-10 ng)

- Sypro Ruby fluorescent stain : • subnanogram detection • good linear range

- Amido black (PVDF) - Ponceau S (PVDF)

- Commerically available detection solution, i.e. Bio-Rad Immun-blot kit. * Nitrocellulose is recommended for use to replace PVDF membrane.

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Imaging software

_{Amersham Pharmacia Biotech}

Typhoon

BIO-RAD

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Imaging software

* ImagemasterTM _{(APB), PDQuest}TM _{(BIO-RAD) and Z3 (Compugen).}

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2D Gel databases

* EXPASY http://www.expasy.ch/ch2d/2d-index.html or http://tw.expasy.ch/ch2d/2d-index.html - Multi species - Mammalia - Yeast - Plant - Bacteria, Viruses & Parasites - Cell lines

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In

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Gel digestion

* Spot picking (gel excision) – manual or mechanical ?

BIO-RAD PROTEAN@

Spot cutter

Amersham Pharmacia Biotech

Ettan Spot picker

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In

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Gel digestion

1. All tubes and tips are washed with methanol, rinsed with Milli-Q water and methanol, then dried.

2. Trim the excised gel into small pieces (1 mm3_{). Add 120 µl of wash solution}

(50% v/v acetonitrile in 25 mM ammonium bicarbonate, pH 7.8) to destain color. 3. Shake the tube/plate at 37 º C for 10 min and drain the solution.

4. Repeat step 2 & 3 until no blue color is visible. 5. Speedvac the gel pieces for 15 min to dry.

6. Add 8 µl (15 ng/µl) sequencing grade trypsin (in 25 mM NH₄HCO₃, pH 7.8) to gel sample.

7. Incubate at 37ºC for at least 16 hr.

8. Spin tube/plate to concentrate liquid on bottom of tube/well. 9. Add 8 µl extract solution ((50% v/v acetonitrile, 1% v/v TFA) 10. Sonicate for 20 min in water bath sonicator.

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What is the next?

* Sample preparation for mass spectrometry: - MALDI-TOF (1 µl) - ESI-Q-TOF (5-10 µl)