Regardless of how much protein is applied to the membrane, it must be retained at the point of application to contribute to the signal. Although the capture reagent is localized at the point of application simply as a consequence of evaporation of the water, whether it remains in place when the sample is run depends on how tightly it is adsorbed to the nitrocellulose (10,11). The effectiveness of adsorption is in turn a function of the other solutes used in the reagent buffer and their interaction with the nitrocellulose and capture reagent. Chemistries used to preserve biological activity can be detrimental to adsorption. Surfactants, such as Tween-20, commonly used to prevent aggregation, interfere with adsorption by preventing the protein from coming into contact with the nitrocellulose at the molecular level (10,11). Also, water can interfere with adsorption when not completely evaporated after reagent application. Albumin conjugates may require more aggressive desiccation than antibodies.
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