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Protein Visualization

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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Protein Visualization

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  1. Protein Visualization 潘台龍博士 長庚大學中醫系

  2. Sensitivity Biological Function Quantification Throughput Diverse Challenges in Proteomics……….. Bioinformatics

  3. 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

  4. 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,

  5. 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)

  6. 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).

  7. Protein Dyes 30 - 100ng 8 - 50ng

  8. SYPRO Ruby stained gel Silver stained gel C B B C A A D D E. coli lysate

  9. 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)

  10. Gel Doc 2000 Fluor-S Molecular Imager FX

  11. 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

  12. 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

  13. 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

  14. 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

  15. Ammoniacal Silver Staining • Fixation • Sensitization • Silver impregnation • Image development • Stopping development and image stabilization Problems: High backgrounds, reproducibility, silver mirror thiosulfate

  16. 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

  17. 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

  18. SYPRO Ruby Fluorescent Staining Gel with Reagent A Gel with SYPRO Ruby Gel with Reagent B Fluorescence scanner

  19. Fluorescent staining compared with silver staining of 2-DE

  20. 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

  21. Glycoprotein detection of 2-DE separated proteins • Periodic acid/Schiff staining • Digoxigenin (DIG)/AntiDIG alkaline phosphatase (AP) labeling • Lectin

  22. Detection of proteins on membranes Ponceau S CBR250 Amido Black India Ink Colloidal Gold

  23. 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

  24. Fluorescence Difference Gel Electrophoresis

  25. Model System Results Using Cy3 and Cy5 Merged images of: Cy3: control - Blue Cy5: + benzoic acid - Red

  26. Model System Using Cy3 and Cy5 Cy3 Cy5 Ref. Journal of Bacteriology Dec 1997, p.7595-7599

  27. Difference gel electrophoresis (DIGE)

  28. Cy5 labelled gel Silver stained gel E. coli lysate, 50mg loading, pH 4-7 Comparison of Spot Patterns

  29. UNLABELLEDSAMPLE LABELLED SAMPLE Compatible with Mass Spec

  30. Data Revealed by Fluorescent Labelling • Peaks represent individual spots • Single contrast used to display image is incapable of showing fluorescent dynamic range

  31. 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

  32. ICAT Analysis Strategy ICAT- isotope-coded affinity tags

  33. Gygi, 1999

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