User Manual

Blotting
13
13.1 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 Criterion
™
precast gels onto nitrocellulose or PVDF membranes using
either wet or semi-dry transfer techniques.
Assess transfer efficiency using a total protein blot stain such as SYPRO Ruby stain (see Table 12.1).
With Criterion
™
TGX Stain-Free
™
and Criterion Stain Free
™
gels, transfer efficiency to low-fluorescence
PVDF membranes may also be assessed using the Gel Doc
™
EZ imager (see Chapter 5; activate the gel
before blotting).
13.2 Transfer
13.2.1 Transfer Buffers
See Appendix B for buffer formulations. Do not adjust pH unless directed to do so.
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.
13.2.2 Wet Transfer Using the Criterion Blotter
1. Equilibrate the gels and membranes (for example, in Towbin buffer) for 15 min prior to blot assembly.
2. Assemble the transfer apparatus:
a) Fill the tank with transfer buffer to ~50% of the fill volume and place a magnetic stir bar inside
the tank.
b) Place the ice block in the pocket in the back of the cell. Flip down the lever to hold the ice
block. Alternatively, connect the optional cooling coil to an appropriate recirculating water chiller
and place it in the grooves in the back of the tank.
3. Assemble the cassette:
a) Pour chilled transfer buffer into each compartment of the assembly tray, and then place the
membrane (nitrocellulose, PVDF) into the front (small) compartment of the tray to soak.
Wet PVDF membranes in methanol before soaking in transfer buffer.
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