Transcript of CEG Protein Analysis Workshop Two-Dimensional Gel Electrophoresis Yu Liang, Ph.D. Proteomics Core...
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- CEG Protein Analysis Workshop Two-Dimensional Gel
Electrophoresis Yu Liang, Ph.D. Proteomics Core Facility at UC
Medical Center
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- 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 post- translational modifications (PTM)
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- Proteomics Overview Sample Preparation2-D Electrophoresis Spot
Detection & Image Analysis Enzymatic DigestionPeptide-Mass
Fingerprinting Peptide Sequencing via MSDatabase Search Protein
Identification (I)(III) (IV)(V)(VI) (II)
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- Personnel 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
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- 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)
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- Genomic Solutions 2D Electrophoresis System pH phaser
isoelectric focusing system Investigator 2-D electrophoresis
running system
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- 2-D Image (Fluorescent Staining) mouse cardiac 250 g loading pH
3-10 IEF strips 12.5% SDS-PAGE File ID: mb699
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- 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
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- Sample gels ControlExperimental
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- 2-D Image Control mouse cardiac; 250 g loading; pH 3-10 IEF
strips; 12.5% SDS-PAGE; file ID: sc13con vs. sc08iso; spot ID: S8-1
Experimental
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- 2-D Image mouse cardiac; 250 g loading; pH 3-10 IEF strips;
12.5% SDS-PAGE; file ID: sc13con vs. sc08iso Control Experimental
Spot ID: S8-1
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- 2-D Image mouse cardiac; 250 g loading; pH 3-10 IEF strips;
12.5% SDS-PAGE; file ID: sc5bcon vs. sc15iso Control Experimental
PTM? Downregulation?
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- 2-D Gel Analysis
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- 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
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- www.med.uc.edu/proteomics
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- Contact Protocols Price list Services News & Events
Feedback Publications On-line order FAQs
www.med.uc.edu/proteomics
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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? FAQs
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- 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.
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- 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 )
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- 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
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- 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 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
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- Blue: Pro-Q Diamond phosphoprotein gel stain Red: SYPRO Ruby
total protein stain (Molecular Probes Website)
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- Samples Wanted Among 0.5~1 million proteins in human proteome,
whats your favorite one? We are here to help the hunting. Please
send your samples to: Proteomics Core MSB5301 Tel: 558-2347
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- Special Thanks Dr. John Maggio Dr. Limbachs MS Core Dr. Stephen
Macha http://www.chembus.uc.edu/massnew/maintbl.asp