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Protein Visualization . 潘台龍博士. 長庚大學中醫系. Sensitivity. Biological Function. Quantification. Throughput. Diverse Challenges in Proteomics………. Bioinformatics. Stain techniques . General comments. Techniques - The chemistry of all stains is to bind a chromaphore to a polypeptide

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slide1

Protein Visualization

潘台龍博士

長庚大學中醫系

slide3

Sensitivity

Biological

Function

Quantification

Throughput

Diverse Challenges in Proteomics………..

Bioinformatics

stain techniques
Stain techniques

General comments

  • Techniques - The chemistry of all stains is to bind a chromaphore to a polypeptide
  • Quantification - All stains are very qualitative, individual polypeptides differ greatly in their ability to bind a stain
  • Reproducibility - Different staining techniques will not stain a polypeptide consistently
  • Range - There is a small concentration range over which a particular stains is useful
slide5

General protein detection methods

  • Organic dye- and silver stain-based methods
  • Radioactive labeling methods
  • Reverse stain methods
  • Fluorescence-based staining methods
  • Chemiluminescence-based staining methods
  • Immobilized pH gradient /MALDI-TOF mass spectrometry and surface-enhanced laser desorption–ionization mass spectrometry (SELDI-MS)

Specific detection of protein post-translational modifications

  • Glycoprotein detection methods
  • Phosphoprotein detection methods
  • Proteolytic modification detection methods
  • S-Nitrosylation detection method
  • Arginine methylation detection methods
  • ADP-ribosylation detection methods

771 (2002) 3–31 Journal of Chromatography B,

slide6

Detection of reporter enzymes and epitope tags

  • b-Glucuronidase
  • b-Galactosidase
  • Oligohistidine tags
  • Green fluorescent protein

Differential display proteomics: approaches and options

  • Difference gel electrophoresis (DIGE)
  • Multiplexed proteomics (MP)
  • Isotope-coded affinity tagging (ICAT)
slide7

Noncovalent interaction

  • Organic dyes- Coomassie brilliant blue and amidoblack. Detection is by light absorption
  • Fluorescent probes- Sypro dyes and Nile red. Detection is by fluorescence.
  • Metal ion binding
  • - salt binding (negative or reverse staining with zinc-imidazole).
  • -metal ion reduction (silver stain).
protein dyes
Protein Dyes

30 - 100ng

8 - 50ng

slide9

SYPRO Ruby stained gel

Silver stained gel

C

B

B

C

A

A

D

D

E. coli lysate

slide10

Imaging Systems

GS-710 DensitometerCalibratedReflectance and Transmittance

Fluor-S / Fluor-S MaxMultiImager CCD Based UV / Visible Fluorescence& Chemiluminescence

Molecular Imager FX Laser-Based Scanner Fluorescence and Storage Phosphor Imaging488 nm, 532 nm (635 nm)

slide11

Gel Doc 2000

Fluor-S

Molecular Imager FX

image analysis with typhoon
Image Analysis with Typhoon
  • Multi-mode
    • Phosphorimaging
    • Fluorescence (4 dyes per sample)
    • Chemiluminescence
  • Flexibility
    • Sample size up to 35x43 cm
    • Use gels within glass plates
    • Up to 17 filters
  • High Sensitivity
    • In the attomole range for fluorescence
  • 5 orders of magnitude and 16 bits
  • High resolution
    • Up to 50 microns
  • Linked to ImageMaster SW
image analysis with imagescanner
Image Analysis with ImageScanner
  • Flexible
    • For small and large gels
    • Transmission and reflection mode
    • Selection of scan colour
  • High dynamic range
    • 0.00 to >3.4 O.D.in 16384 steps
  • High resolution
    • Up to 10 microns
  • Fast
    • Scans a large gel in 40 sec.
  • Linked to ImageMaster SW
slide14

Conventional Coomassie Blue

  • Coomassie Brillant Blue 250 (CRB-250)-complex with basic amino acids.Q:_
  • Coomassie Brilliant Blue R250

500 ml methanol

100 ml Acetic acid

400 ml H2O

1 g CBR250

  • Destain

810 ml H2O

120 ml methanol

70 ml Acetic acid

slide15

Colloidal Coomassie Staining

(10% Acetic acid/40%Methanol)

Fix the gel

Wash the gel with H2O

Stain the gel with colloidal Coomassie stain

50g (NH4)2SO4

6 ml 85% phosphoric acid

10 ml 5% CBG250

H2O 500 ml

Wash the gel with H2O

slide16

Ammoniacal Silver Staining

  • Fixation
  • Sensitization
  • Silver impregnation
  • Image development
  • Stopping development and image stabilization

Problems:

High backgrounds, reproducibility, silver mirror

thiosulfate

slide17

Mass-spectrometry-compatible silver staining

Gel with Reagent A

600 ml ethanol

200 ml acetic acid

1200 ml H2O

Gel with Reagent B

6g potassium tetrathionate

98g potassium acetate

/600 ml ethanol

1200 ml H2O

Wash Gel with H2O

Gel with Reagent C

2g silver nitrate

1200 ml H2O

Wash Gel with H2O

60g potassium carbonate

600ul formaldehyde

250ul 10% sodium thiosulfate pentahydrate/2000 ml H2O

Gel with Reagent D

80g Tris & 40 ml Acetic acid/2000 ml H2O

Gel with Reagent E

slide18

Imidazole-Zinc Negative Staining Reagent

Gel with 1% sodium carbonate

Gel with imidazole-SDS soln’s

Wash Gel with H2O

Gel with zinc acetate soln’s

slide19

SYPRO Ruby Fluorescent Staining

Gel with Reagent A

Gel with SYPRO Ruby

Gel with Reagent B

Fluorescence scanner

slide21

Phosphoprotein Staining with the GelCode Phosphoprotein Staining kit

Gel with H2O

Gel with Sulfosalicylic acid

Gel with H2O

Gel with 0.5N NaOH

Gel with ammonium molybdate soln’s

Gel with methyl Green soln’s/sulfosalicylic acid

slide22

Glycoprotein detection of 2-DE separated proteins

  • Periodic acid/Schiff staining
  • Digoxigenin (DIG)/AntiDIG alkaline phosphatase (AP) labeling
  • Lectin
slide23

Detection of proteins on membranes

Ponceau S

CBR250

Amido Black

India Ink

Colloidal Gold

proteomics ta programme
Proteomics TA Programme
  • Proteomics TA is based on fluorescent 2D DIGE technology and is an integral part of the overall Proteomics strategy
    • Initial programme gives access to fluorescent 2D DIGEtechnology, Cyanine dyes (CyDye™) Cy 2, Cy 3, Cy 5, matching hardware, software and consumables
    • Optional modules for 2D DIGE MS will be launched in a modular fashion in 2001, including spot picker, digestion station and spotter modules specific for TA
slide26

Model System Results Using Cy3 and Cy5

Merged images of:

Cy3: control - Blue

Cy5: + benzoic acid - Red

slide27

Model System Using Cy3 and Cy5

Cy3 Cy5

Ref. Journal of Bacteriology Dec 1997, p.7595-7599

comparison of spot patterns

Cy5 labelled gel

Silver stained gel

E. coli lysate, 50mg loading, pH 4-7

Comparison of Spot Patterns
data revealed by fluorescent labelling
Data Revealed by Fluorescent Labelling
  • Peaks represent individual spots
  • Single contrast used to display image is incapable of showing fluorescent dynamic range
proteomics ta programme1
Proteomics TA Programme
  • Initial Programme Contents, Level 1
    • Access to the Cyanine dye chemistry
    • Minimal & Saturation Dyes
    • Licence to the 2D DIGE technology
    • Proprietary Imager, matched to dyes
    • ImageMaster™ 2D Software
    • Special DeCyder S/W
    • IPGPhor™ electrophoretic 1st dimension hardware
    • Training course and privileged R&D support
    • Service offering
slide33

ICAT Analysis Strategy

ICAT- isotope-coded affinity tags