Biology Reference
In-Depth Information
2. Materials
The 2DE equipment employed was an Ettan DALT twelve System
from GE Healthcare (Little Chalfont, Buckinghamshire, UK)
and most of the associated hardware, consumables and manuals/
protocols were from the same supplier.
2.1. Equipment
1. Pestle and mortar.
2. Ice bath.
3. Spectrophotometer.
2.1.1. Protein Extraction
from Mouse Liver Tissue
1. Ice bath.
2. Glass syringe, 500 mL.
2.1.2. Protein Labelling
1. IPG strips (3-10NL, 24 cm; GE Healthcare).
2. Rehydration tray and lid (GE Healthcare).
2.1.3. Rehydration
of IPG Strips
1. Ettan IPGphor II with electrodes (GE Healthcare).
2. IEF ceramic tray and wicks (GE Healthcare).
3. Two pairs of tweezers.
4. Spirit level.
2.1.4. Isoelectric Focusing
1. Rehydration tray and lid (GE Healthcare).
2. Two pairs of tweezers.
3. Thin but blunt long-bladed spatula.
4. Microwave oven.
5. Ettan DALT twelve separation unit (GE Healthcare).
6. Rehydration tray and lid (GE Healthcare).
2.1.5. Second-Dimension
Gel Electrophoresis
1. Typhoon 9400 plus blue laser module and control software
(GE Healthcare).
2. Gel alignment guide (GE Healthcare).
3. ImageQuant v5 software (GE Healthcare).
4. Progenesis SameSpots (Non-Linear Dynamics, Newcastle upon
Tyne, UK).
2.1.6. Gel Scanning
and Image Analysis
1. Staining trays.
2. Light box.
3. Manual spot picker (e.g. One Touch Spot Picker, available from
the Gel Company) and compatible tips with 1.5 mm-orifi ce.
4. Fine nose tweezers.
2.1.7. Visualisation
and Picking of Protein
Spots of Interest
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