Course Manual

  1. Dr. Reiner Westermeier,
  2. Dr. Tom Naven

Published Online: 7 MAY 2002

DOI: 10.1002/3527600175.ch3

Book Title

Proteomics in Practice: A Laboratory Manual of Proteome Analysis

Proteomics in Practice: A Laboratory Manual of Proteome Analysis

Additional Information

How to Cite

Westermeier, R. and Naven, T. (2002) Course Manual, in Proteomics in Practice: A Laboratory Manual of Proteome Analysis, Wiley-VCH Verlag GmbH, Weinheim, FRG. doi: 10.1002/3527600175.ch3

Author Information

  1. Amershan Biosciences Europe GmbH, Munzinger Str. 9, 79111 Freiburg, Germany

Publication History

  1. Published Online: 7 MAY 2002
  2. Published Print: 9 MAY 2002

ISBN Information

Print ISBN: 9783527303540

Online ISBN: 9783527600175

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CHAPTER TOOLS

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Keywords:

  • stock solution;
  • microdialysis;
  • precipitation;
  • SDS samples;
  • HMW proteins separation;
  • isoelectric focusing;
  • reswelling tray;
  • IEF;
  • IPGphor strip holder;
  • cup loading IEP;
  • staining of IPG strips;
  • SDS polyacrylamide gel electrophoresis;
  • Coomassie Brilliant Blue staining;
  • silver staining;
  • scanning of gels;
  • spot detection;
  • 2-D patterns;
  • fluorescence difference gel electrophoresis;
  • CyDye labelling;
  • spot excision;
  • sample destaining;
  • in-gel digestion;
  • microscale desalting;
  • MS analysis;
  • MALDI-ToF MS calibration

Summary

  • Equipment, Consumables, Reagents

  • Step 1: Sample preparation

    • Stock solution

    • Examples

    • Microdialysis

    • Precipitation

    • Basic proteins

    • Very hydrophobic proteins

    • Quantification

    • SDS samples for HMW proteins separation

  • Step 2: Isoelectric focusing

    • Reswelling tray

    • Rehydration loading and IEF in IPGphor strip holder

    • IEF in the cup loading strip holder (rehydration loaded strips)

    • Cup loading IEP

    • Staining of IPG strips

  • Step 3: SDS Polyacrylamide Gel Electrophoresis

    • Casting of SDS polyacrylamide gels

    • Inserting ready-made gels into cassettes

    • Preparation of the SDS electrophoresis equipment

    • Stock solutions for the running buffers

    • Setting up the integrated high-throughput instrument

    • Setting up the six gel instrument

    • Equilibrium of the IPG strips onto SDS gels

    • The SDS electrophoresis run

  • Step 4: Staining of the gels

    • Colloidal Coomassie Brilliant Blue staining

    • Hot Coomassie Brilliant Blue staining

    • Silver staining

    • Preserving and dying of gels

  • Step 5: Scanning of gels and image analysis

    • Scanning

    • Spot detection and background parameters

    • Evaluation of 2-D patterns

  • Step 6: Fluorescence difference gel electrophoresis

    • Preparing a cell lysate compatible with CyDye labelling

    • Reconstituting the stock CyDye in Dimethylformamide (DMF)

    • Preparing CyDye solution used to label proteins

  • Step 7: Spot excision

  • Step 8: Sample destaining

  • Step 9: In-gel digestion

  • Step 10: Microscale desalting and concentration of sample

  • Step 11: Chemical derivatisation of the peptide digest

  • Step 12: MS analysis

  • Step 13: Calibration of the MALDI-ToF MS

  • Step 14: Preparing for a database search

  • Step 15: PMF database search unsuccessful

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