Does Setting the Shaker to 130 rpm During the Blocking Step in Western Blot Compromise Experimental Outcomes?
In Western blotting, an unintended setting of 130 rpm for orbital shaking during the blocking step is generally not expected to compromise the quality or interpretability of the results. The primary aim of blocking is to reduce nonspecific binding of antibodies, which largely depends on the choice and concentration of the blocking reagent, as well as the blocking duration.
Shaking during this step serves to facilitate uniform distribution of the blocking buffer across the membrane surface, thereby preventing localized drying or incomplete coverage. In most protocols, minor deviations in shaking speed do not substantially affect blocking efficiency. However, excessively high speeds can pose risks such as membrane damage or sample displacement, while very low speeds may result in uneven blocking. A speed of 130 rpm is typically considered moderate and unlikely to negatively influence the blocking performance.
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