flag

Methods


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