Professional Documents
Culture Documents
- 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
40 mM Tris Supernatant 1
Insoluble pellet 2
5M urea, 2M thiourea, 2% CHAPS,
2% SB 3-10, 2mM TBP, highly hydrophobic
0.2% ampholytes, 40 mM Tris
Supernatant 3
* Modified from APAF protocol
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
Multiphor II
Two-Dimensional Gel Electrophoresis (2-DGE)
- Sample loading on IPG gels
* Protein loadings for gels (guide only)
BIO-RAD
Molecular Imager FX
Two-Dimensional Gel Electrophoresis (2-DGE)
- Imaging software
* ImagemasterTM (APB), PDQuestTM (BIO-RAD) and Z3 (Compugen).
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