A new discontinuous buffer system using a Tris-CAPS buffer provides excellent results in semi-dry blotting. This new procedure uses Tris-CAPS buffer plus 15% methanol in the filter paper on the anode and Tris-CAPS plus 0.1% SDS in the filter paper on the cathode.
Cytosol from white blood cells was separated by SDS-PAGE. The phox-47 component of the Respiratory Burst Oxidase was detected by Immun-Star chemiluminescent substrate.
For Best Results
Bio-Rads Extra Thick Blotting Paper should be used. This paper is 2.6 mm thick, 100% cotton fiber, and provides the absorbency required for semi-dry blotting.
PVDF provides superior retention of the protein. For proteins less than 20 K it is required to prevent blow-through during electrophoretic transfer. Its higher binding capacity will increase detection results. It is important to first wet the PVDF in 100% methanol, then equilibrate in bottom/anode buffer for at least 30 minutes prior to transfer. It is expedient to begin equilibration during the vertical electrophoresis run.
Tris-CAPS (60 mM Tris base, 40 mM CAPS, pH 9.6)
5x Stock Solution
36.34 g Tris-base
44.26 g CAPS
Water to 1 liter
To prepare 100 ml working buffers from 5x stock solution:
Bottom/Anode buffer: Top/Cathode bu