Sheet Protector Strategy for Western Blot to Reduce Antibody Consumption and Incubation Time.

Abstract

Background: Western blot is one of the most routinely conducted biochemical assays due to its technical ease and relatively low cost. The use of antibody is at the center of Western blot assay, providing great sensitivity and specificity. However, challenges can be posed when using a rare antibody stock. There have been efforts to improve the Western blot procedure to minimize the use of antibody, but these methods require specialized devices.

Results: In this study, we hypothesized that the conventional large pool of antibody is not essential for detection and attempted applying only a small volume of antibody. We used sheet protector (SP), a common stationery material that protects office documents, to effectively distribute the antibody solution over the nitrocellulose (NC) membrane. This way, 20-150 µL antibody solution was sufficient for mini-sized membrane, which was adjustable depending on the size of the membrane. We confirmed that the sensitivity and specificity of this SP strategy was comparable to conventional (CV) method. The SP strategy brought a few additional advantages including: (1) antibody incubation without agitation (2), incubation at room temperature, and (3) faster detection on the order of minutes. Finally, we examined 15-min incubation SP protocol using time-series apoptosis samples.

Conclusions: We propose that SP strategy is a universally accessible approach using a common consumable, greatly enhancing the efficiency of antibody consumption and incubation time in Western blot assays.