( transfer because the buffer reservoirs...)Increased Transfer Efficiency Using a Discontinuous Buffer System With the Trans-Blot SD Cell

transfer because the buffer reservoirs, the filter paper on both sides of the gel, are independent. In a discontinuous system, methanol should be included in the buffer on the membrane side (anode) of the blot assembly and SDS used on the gel side (cathode), taking advantage of the positive effects of each component.

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.

Buffer Formulation
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
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