Two-Dimensional Gel Electrophoresis (2-DGE)
- 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
Two-Dimensional Gel Electrophoresis (2-DGE)
- 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
Two-Dimensional Gel Electrophoresis (2-DGE)
- 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
Two-Dimensional Gel Electrophoresis (2-DGE)
- 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
Two-Dimensional Gel Electrophoresis (2-DGE)
- 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
Two-Dimensional Gel Electrophoresis (2-DGE)
- 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
O H2N
Urea
CH2
NH2
S
H2N
NH2
Thiourea
R (CH2)3 NCH2CH2CH2SO3
CH3 CHAPS
Sulfobetaine
HS
H
H
C
C
OH OH
DTT
SH
Two-Dimensional Gel Electrophoresis (2-DGE)
- Sample preparation
Sequential extraction of proteins:
- decreasing spot numbers, yet still display all the proteins.
SAMPLE
hydrophilic
Supernatant 1
40 mM Tris
Insoluble pellet 1
hydrophobic
8M urea, 4% CHAPS, 2mM TBP,
0.2% ampholytes, 40 mM Tris
Insoluble pellet 2
5M urea, 2M thiourea, 2% CHAPS,
2% SB 3-10, 2mM TBP,
0.2% ampholytes, 40 mM Tris
* Modified from APAF protocol
Supernatant 2
highly hydrophobic
Supernatant 3
Two-Dimensional Gel Electrophoresis (2-DGE)
- 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
Two-Dimensional Gel Electrophoresis (2-DGE)
- Sample loading on IPG gels
* Choice of first-dimension IPG strips
Amersham Pharmacia Biotech
IPGphor
BIO-RAD PROTEAN IEF cell
Multiphor II
Two-Dimensional Gel Electrophoresis (2-DGE)
- Sample loading on IPG gels
* Protein loadings for gels (guide only)
Analytical load (silver or sypro) mini gel
10-50 µg total protein
Analytical load (silver or sypro) large gel
100-200 µg total protein
Preparative load (comassie) mini gel
200-500 µg total protein
Preparative load (comassie) large gel
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.
Two-Dimensional Gel Electrophoresis (2-DGE)
- 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.
Two-Dimensional Gel Electrophoresis (2-DGE)
- SDS-PAGE gels
* IPG equilibration & casting IPG on the second dimension.
REAGENTS
FUNCTION
AMOUNT/FINAL CONC.
Urea
Denaturation and
solubilization of proteins
36g urea (6M)
SDS
Solubilization of proteins
2 g (2%)
5x Tris/HCl gel buffer
Buffering (pH 8.8)
20 ml (1x)
50% glycerol
Inhibits electroendosmosis
in the viscosity stops water
transfer across the IPG
40 ml (20%)
200 mM TBP
Reduction (breaks the
disulfide bonds)
2.5 ml (5mM)
25% Acrylamide soln.
Alkylation (prevents
bonds rejoining,
compatibility for MS)
10 ml (2.5%)
Final volume 100 ml
Two-Dimensional Gel Electrophoresis (2-DGE)
- SDS-PAGE gels
* Casting IPG on the second dimension.
Two-Dimensional Gel Electrophoresis (2-DGE)
- 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.
Two-Dimensional Gel Electrophoresis (2-DGE)
- Imaging software
BIO-RAD
Molecular Imager FX
Amersham Pharmacia Biotech
Typhoon
Two-Dimensional Gel Electrophoresis (2-DGE)
- Imaging software
* ImagemasterTM (APB), PDQuestTM (BIO-RAD) and Z3 (Compugen).
50% decrease
50% increase
Two-Dimensional Gel Electrophoresis (2-DGE)
- 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
Two-Dimensional Gel Electrophoresis (2-DGE)
- In-Gel digestion
* Spot picking (gel excision) – manual or mechanical ?
Amersham Pharmacia Biotech
Ettan Spot picker
BIO-RAD PROTEAN@
Spot cutter
Two-Dimensional Gel Electrophoresis (2-DGE)
- 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 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.
11. Desalt and concentrate with ZipTip (Millipore).
Two-Dimensional Gel Electrophoresis (2-DGE)
- What is the next?
* Sample preparation for
mass spectrometry:
- MALDI-TOF (1 µl)
- ESI-Q-TOF (5-10 µl)
Two-Dimensional Gel Electrophoresis (2-DGE)
- Additional information
* Amersham Pharmacia Biotech - http://www.apbiotech.com.tw/
* BIO-RAD - http://www.proteomeworks.bio-rad.com/
* Millipore - http://www.millipore.com/catalogue.nsf/docs/C5737
* 2-D Electrophoresis: USING IMMOBILIZED PH GRADIENT; PRINCIPLE &
METHODS,
APB (pdf files).
* http://www.yahoo.com
* http://www.ncbi.nlm.nih.gov/PubMed/
Two-Dimensional Gel Electrophoresis (2-DGE)