CEG Protein Analysis Workshop
Two-Dimensional Gel Electrophoresis
Yu Liang, Ph.D.
Proteomics Core Facility at UC Medical Center
From Genotype to Phenotype
Genome: DNAs
 Transcriptome: RNAs
 Proteome: Proteins
 Physiome: Metabolites
 Biome: Environment
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Beyond the Genome
Proteins are ultimately responsible for
all biological processes that take
place within cells.
 Protein dynamics reflect the state of
biological system at a given time
 Detection and identification of posttranslational modifications (PTM)
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Proteomics Overview
Sample Preparation
(I)
Enzymatic Digestion
2-D Electrophoresis Spot Detection & Image Analysis
(II)
Peptide-Mass Fingerprinting Protein Identification
Peptide Sequencing via MS
(IV)
(III)
(V)
Database Search
(VI)
Personnel
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Yu Liang, Ph.D.
Research Associate
MSB 5301
Tel: 558-2347
liangyu@ucmail.uc.edu
http://www.med.uc.edu/proteomics/
John Maggio, Ph.D.
Professor and Chair
Department of Pharmacology & Cell Biophysics
Proteomics Core Equipment
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Genomic Solutions Investigator 2-D
Electrophoresis System
Genomic Solutions Gel Casting System
Genomic Solutions ProImage Image Acquisition
System
Dell Optiplex Image Analysis Computers
New:
Fuji Fluorescent Image Analyzer FLA5100
(Phosphoprotein staining and DIGE)
Genomic Solutions 2D Electrophoresis
System
pH phaser isoelectric focusing
system
Investigator 2-D electrophoresis
running system
2-D Image (Fluorescent Staining)
• mouse cardiac
• 250 g loading
• pH 3-10 IEF strips
• 12.5% SDS-PAGE
• File ID: mb699
Customized Core Services
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Complete 2-D Gel Service with Spot Picking for MS Analysis
Complete 2-D Gel Service, or
(1) IEF Only
(2) SDS-PAGE (large format) Only
(3) SDS-PAGE Plus (staining & imaging)
(4) Gel Staining (fluorescent, Sypro Ruby)
(5) Image Analysis Only
(6) Image Analysis Plus (spot pick)
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Two new services:
(1) Phosphoprotein staining by Pro-Q Diamond
fluorescent dye
(2) Difference gel electrophoresis (DIGE) by Cydye
labelling
Sample gels
Control
Experimental
2-D Image
Control
Experimental
• mouse cardiac; 250 g loading; pH 3-10 IEF strips; 12.5% SDS-PAGE; file ID: sc13con vs. sc08iso; spot ID: S8-1
2-D Image
Control
Experimental
Spot ID: S8-1
• mouse cardiac; 250 g loading; pH 3-10 IEF strips; 12.5% SDS-PAGE; file ID: sc13con vs. sc08iso
2-D Image
Control
PTM?
Downregulation?
Experimental
• mouse cardiac; 250 g loading; pH 3-10 IEF strips; 12.5% SDS-PAGE; file ID: sc5bcon vs. sc15iso
2-D Gel Analysis
2-D Gel Analysis
2-D Gel Analysis
Deliverables
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Fluorescence-stained 2-D gels
Electronic image files
Spot list
Normalized volumes of spots
Plus
 Free access to our software for further analysis
 Free advice and consultation on further protein
characterization as well as 2-D image analysis
www.med.uc.edu/proteomics
www.med.uc.edu/proteomics
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Contact
Protocols
Price list
Services
News & Events
Feedback
Publications
On-line order
FAQs
FAQs
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How do I get started?
 What do I get for my result?
 How much protein should I load for the 2-D gel
analysis?
 How long will it take to get the results?
 Who is responsible for preparation of protein
samples?
 What protocol should I use for protein
solubilization?
Sample Preparation
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The most important step towards the success of 2-D
electrophoresis
Basic rules: keep everything simple
use high-purity reagents
Keep proteins denatured and in solution: 7M urea, 2M thiourea,
and 4% CHAPS.
Break disulfide bonds: 20 mM DTT. (NOT 2-ME)
Prevent protein modification: protease inhibitors; phosphatase
inhibitors.
NO ionic detergent, esp. SDS. Non-ionic detergents are OK, e.g.
Triton X-100 and NP-40.
Keep salt concentration below 10 mM.
Clean up interfering substance, including salt, nucleic acids, lipids,
and polysaccharides: acetone (TCA) precipitation and commercial
kits.
New Fuji FLA 5100
Pro-Q diamond fluorescent staining
for phosphoproteins
 Multiplexing using DIGE
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Multiplexing by Difference Gel Electrophoresis
(DIGE)
DIGE with CyDye labeling of protein
(Amersham Web Site)
Multiplexing by Difference Gel Electrophoresis
(DIGE)
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Comparison of samples within the same gel
eliminates gel-to-gel variation
 Including the internal standards improves
statistical confidence of comparing samples for
protein abundance changes
 Reduces number of gels needed to be run in the
experiment:
2 comparing groups with 6 individuals,
36 gels for regular 2-D gels
12 gels for 2-dye DIGE
6 gels for 3-dye DIGE
Post-Translational Modifications
Important for biological processes,
particularly signal transductions
 Most cellular processes are regulated
by reversible phosphorylation of
proteins
 Studies of phosphorylated proteins
involve radioisotope-labeling and
specific antibodies
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Pro-Q Diamond Staining of a 2-D gel
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Selectively stains phosphoproteins in 2-D gel
 Allows direct in-gel detection of phosphate groups
attached to tyrosine, serine, or threonine residues
 NO NEED for antibody and radioisotope
 Signal correlates with the number of phosphate
groups
 Fully compatible with mass spectrometry
 Ratio of Pro-Q diamond to Sypro Ruby
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=phosphorylation level/total protein
Blue: Pro-Q Diamond
phosphoprotein gel stain
Red: SYPRO Ruby total protein
stain
(Molecular Probes Website)
Samples Wanted…
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Among 0.5~1 million proteins in human
proteome, what’s your favorite one?
We are here to help the hunting.
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Please send your samples to:
Proteomics Core
MSB5301
Tel: 558-2347
Special Thanks…
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Dr. John Maggio
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Dr. Limbach’s MS Core
Dr. Stephen Macha
http://www.chembus.uc.edu/massnew/maintbl.asp