1. Protein purification and separation
2. Protein digestion for MS analysis
3. Liquid-chromatography-based approaches for peptide preparation
4. Mass spectrometry using the LTQ-Orbitrap XL/ETD instrument
5. Biocomputing analysis in proteomics For suggestions on how to prepare your biological material before a proteomics flow can start see:
Preparation of cell lysates
Sample Prep Upstream of Mass Spectrometry 1. Protein purification and separation
1.1 Gel-based approaches
1.1.1 Protein separation by electrophoresis
resources: Basic Primer (Thermo)
2D electrophoresis (MIAPE GE) (Guidelines for reporting the use of gel electrophoresis in proteomics)
- Sample Preparation
- 1st dimension: Isoelectric focusing using the IPGPhor aparratus
resources: IEF strip selector: GE Biorad
- 2nd dimension: SDS/Tricine PAGE (mini and large gels)
resources
SDS-PAGE
resources:
Tips for SDS-PAGE Gel Handling-Fred Hutchinson Cancer Research Center
Native PAGE
resources: Native PAGE theory (NationalDiagnostics) Native Gels (AllianceProteinLabs) Protocol
Blue-Native PAGE
resources: CSHLprotocols Eubel2005(Review)
Preparative SDS or Native PAGE using the BioRad prep-cell 491- manuals
Hydrophobic proteins
resources: Lin1999(Review)
1.1.2 Protein staining
Protein fixing
resources: Fixing (NationalDiagnostics)
CoomassieBlue staining
"Blue Silver" Staining (MS-compatible)
Silver Staining
Silver Staining (MS-compatible)
resources: Make the Amersham Plus-One silver stain kit (17-1150-01) MS-compatible: pdf (4K)
1.1.3 Gel scanning
resources: guide (Decodon)
1.1.4 Gel imaging
Analysis of 2D gels using Delta2D software
1.2 Gel-free approaches
1.2.1 Liquid chromatography (FPLC/HPLC)
Size exclusion chromatography
resources: GelFiltration manual(GE)
Ion exchange chromatography (SCX, Q)
Affinity chromatography HisEspA-HisCesAB purification Native conditions
Reversed-phase chromatography (C4, C8, C18)
Multi-dimensional LC (different combinations of the above)
1.2.2 Preparative Isoelectric focusing
Using the Rotophor apparatus
1.2.3 Capilary Electrophoresis
resources: CE-MS
1.2.4 Sedimentation-based separation
resources: RateZonalSedimentationInTheOptimaTLX Proteomic Complex Detection Using Sedimentation
1.2.5 Preparation of membrane proteins
1.2.6 Ultrafiltration
resources: HighthroughputUltrafiltration
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