1. CEG Protein Analysis Workshop
Two-Dimensional Gel Electrophoresis
Yu Liang, Ph.D.
Proteomics Core Facility at UC Medical Center

2. From Genotype to Phenotype
Genome: DNAs
Transcriptome: RNAs
Proteome: Proteins
Physiome: Metabolites
Biome: Environment

3. 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 post-translational modifications (PTM)

4. Proteomics Overview (I) (II) (III) (IV) (V) (VI) Sample Preparation
2-D Electrophoresis
Spot Detection & Image Analysis
(I)
(II)
(III)
Enzymatic Digestion
Peptide-Mass Fingerprinting
Protein Identification
Peptide Sequencing via MS
Database Search
(IV)
(V)
(VI)

5. Personnel Yu Liang, Ph.D. Research Associate MSB 5301 Tel: 558-2347
John Maggio, Ph.D.
Professor and Chair
Department of Pharmacology & Cell Biophysics

6. Proteomics Core Equipment
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)

7. Genomic Solutions 2D Electrophoresis System
pH phaser isoelectric focusing
system
Investigator 2-D electrophoresis
running system

8. 2-D Image (Fluorescent Staining)
mouse cardiac
250 g loading
pH 3-10 IEF strips
12.5% SDS-PAGE
File ID: mb699

9. Customized Core Services
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)
Two new services:
(1) Phosphoprotein staining by Pro-Q Diamond
fluorescent dye
(2) Difference gel electrophoresis (DIGE) by Cydye
labelling

10. Sample gels
Control
Experimental

11. 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

12. 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

13. 2-D Image Control Experimental PTM? Downregulation?
mouse cardiac; 250 g loading; pH 3-10 IEF strips; 12.5% SDS-PAGE; file ID: sc5bcon vs. sc15iso

14. 2-D Gel Analysis

15. 2-D Gel Analysis

16. 2-D Gel Analysis

17. Deliverables 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

19. www.med.uc.edu/proteomics Contact Protocols Price list Services
News & Events
Feedback
Publications
On-line order
FAQs

20. FAQs 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?

21. Sample Preparation
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.

22. New Fuji FLA 5100
Pro-Q diamond fluorescent staining for phosphoproteins
Multiplexing using DIGE

23. Multiplexing by Difference Gel Electrophoresis (DIGE)
DIGE with CyDye labeling of protein
(Amersham Web Site)

24. Multiplexing by Difference Gel Electrophoresis (DIGE)
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

25. 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

26. Pro-Q Diamond Staining of a 2-D gel
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
=phosphorylation level/total protein

27. Blue: Pro-Q Diamond phosphoprotein gel stain
Red: SYPRO Ruby total protein stain
(Molecular Probes Website)

28. Samples Wanted…
Among 0.5~1 million proteins in human proteome, what’s your favorite one?
We are here to help the hunting.
Please send your samples to:
Proteomics Core
MSB5301
Tel:

29. Special Thanks… Dr. John Maggio Dr. Limbach’s MS Core
Dr. Stephen Macha