Bio-Rad Mini-PROTEAN® TGX™ Precast Gels for 2-D Electrophoresis - Manual (Page 31)

Blotting
11
11.1 Introduction
Western blotting is an electrophoretic technique used to move proteins from a gel onto a solid support,
such as a nitrocellulose or PVDF membrane. The membrane can be used for immunological or
biochemical analyses or demonstration of protein-protein or protein-ligand interactions.
Below are guidelines for western blotting of Mini-PROTEAN ® precast gels onto nitrocellulose or PVDF
membranes using either wet or semi-dry transfer techniques. After transfer, assess transfer efficiency
using a total protein blot stain (see Section 11.3); with Mini-PROTEAN ® TGX Stain-Free ™ gels, transfer
efficiency to low fluorescence PVDF membranes may also be assessed using the Gel Doc ™ EZ or
ChemiDoc ™ MP imager (see Chapter 5; activate the gel before blotting).
See Appendix B for buffer formulations. Do not adjust pH unless directed to do so.
11.2 Transfer
11.2.1 Transfer Buffers
Towbin buffer (1x)
25 mM Tris, 192 mM glycine, 20% (v/v) methanol (pH 8.3)
Dilute 100 ml 10x stock (catalog #161-0734) with 400 ml diH
2
O.
Add 200 ml methanol, then adjust volume to 1 L with diH
2
O.
Add SDS to 0.1% to promote transfer of high molecular weight proteins.
11.2.2 Wet Transfer Using the Mini Trans-Blot ® Module
1. Equilibrate the gels in transfer buffer for 10–20 min prior to blot assembly.
2. Assemble the Mini Trans-Blot cassette. Place the gel closest to the black plate and the membrane
closest to the red plate of the cassette. Use a roller to remove air trapped between the layers of the
blot assembly.
Wet PVDF membranes in methanol before soaking in transfer buffer.
3. Place the assembled cassette into the transfer module and tank. The red cassette plate should face
the red side of the transfer module. Repeat steps 2 and 3 for a second blot, if needed.
4. Add the cooling unit and stirbar, and fill the tank with transfer buffer. Place the tank on a stir plate,
and begin stirring to maintain even buffer temperature and ion concentration during the transfer.
5. Connect the Mini Trans-Blot cell to a suitable power supply and begin transfer.
For many proteins, excellent transfer efficiency is obtained in 30 min at a constant voltage of 100 V. For
best results, optimize conditions for proteins of interest. Large proteins (>150 kD) may take 60 min, while
smaller proteins (<30 kD) may transfer in 20 min. Refer to the Mini Trans-Blot Instruction Manual (bulletin
1703910) or the Protein Blotting Guide (bulletin 2895) for additional information.
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