Two-Dimensional Gel Electrophoresis (2-DGE)

Transcription

1 - Introduction - Sample preparation - First dimension: Isoelectric focusing - Second dimension: SDS-PAGE - Detection of protein spots: staining - Imaging analysis & 2D Gel databases - Spot handling: excision, in-gel digestion

2 - 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 -pi

3 - Introduction * 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

4 - 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

5 - Sample preparation * A critical step in 2-DE. * Solubilization, denaturation, reduction & removal of non-protein sample components. * Standard sample solubilization solution: * Modified from APAF protocol

6 - 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 Reducing agents: 100mM DTT 2mM TBP (Tributyl phosphine) and/or 65mM DTT O H 2 N NH 2 Urea S H 2 N NH 2 Thiourea R (CH 2 ) 3 CH 2 NCH 2 CH 2 CH 2 SO 3 CH 3 CHAPS Sulfobetaine H H HS C C SH OH OH DTT

7 - Sample preparation Sequential extraction of proteins: - decreasing spot numbers, yet still display all the proteins. SAMPLE hydrophilic 40 mm Tris Supernatant 1 Insoluble pellet 1 8M urea, 4% CHAPS, 2mM TBP, 0.2% ampholytes, 40 mm Tris hydrophobic Supernatant 2 Insoluble pellet 2 * Modified from APAF protocol 5M urea, 2M thiourea, 2% CHAPS, 2% SB 3-10, 2mM TBP, 0.2% ampholytes, 40 mm Tris highly hydrophobic Supernatant 3

8 - 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

9 - Sample loading on IPG gels * Choice of first-dimension IPG strips Amersham Pharmacia Biotech IPGphor BIO-RAD PROTEAN IEF cell Multiphor II

10 - Sample loading on IPG gels * Protein loadings for gels (guide only) Analytical load (silver or sypro) mini gel Analytical load (silver or sypro) large gel Preparative load (comassie) mini gel Preparative load (comassie) large gel µg total protein µg total protein µg total protein 1-3 mg total protein * 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.

11 - 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 high in glycoprotein - purified proteins * Choose available ph range and size IPG (7cm, 11 cm, 18cm, & 24 cm) *Storage at 80ºC.

12 - SDS-PAGE gels * IPG equilibration & casting IPG on the second dimension. REAGENTS Urea SDS 5x Tris/HCl gel buffer 50% glycerol 200 mm TBP 25% Acrylamide soln. FUNCTION Denaturation and solubilization of proteins Solubilization of proteins Buffering (ph 8.8) Inhibits electroendosmosis in the viscosity stops water transfer across the IPG Reduction (breaks the disulfide bonds) Alkylation (prevents bonds rejoining, compatibility for MS) AMOUNT/FINAL CONC. 36g urea (6M) 2 g (2%) 20 ml (1x) 40 ml (20%) 2.5 ml (5mM) 10 ml (2.5%) Final volume 100 ml

13 - SDS-PAGE gels * Casting IPG on the second dimension.

14 - Staining * Sensitive, quantitative and MS-compatible. - Comassie brilliant blue R250 staining ( ng) - Colloidal comassie blue G250 staining ( 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.

15 - Imaging software Amersham Pharmacia Biotech Typhoon BIO-RAD Molecular Imager FX

16 - Imaging software * Imagemaster TM (APB), PDQuest TM (BIO-RAD) and Z3 (Compugen). 50% decrease 50% increase

17 - 2D Gel databases * EXPASY or -Multi species -Mammalia -Yeast -Plant -Bacteria, Viruses & Parasites - Cell lines

18 - In-Gel digestion * Spot picking (gel excision) manual or mechanical? Amersham Pharmacia Biotech Ettan Spot picker BIO-RAD PROTEAN@ Spot cutter

19 - In-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 mm 3 ). 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 NH 4 HCO 3, 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. 11. Desalt and concentrate with ZipTip (Millipore).

20 - What is the next? * Sample preparation for mass spectrometry: - MALDI-TOF (1 µl) -ESI-Q-TOF(5-10 µl)

21 - Additional information * Amersham Pharmacia Biotech - * BIO-RAD - * Millipore - * 2-D Electrophoresis: USING IMMOBILIZED PH GRADIENT; PRINCIPLE & METHODS, APB (pdf files). * *

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