How to measure the affinity of aptamers for membrane proteins expressed on the surface of living adherent cells

• Method to measure the affinity of radioactive aptamers on living adherent cells.
• Optimization of different parameters (incubation, competitors, washings).
• Semi-automation of the binding method using a liquid handling robot.
• Calculation of the apparent Kd and apparent number of targets per cell.
• Guideline for the reporting of binding results.

Recently, an increasing number of aptamers have been selected against biomarkers that are expressed at the surface of cells. This class of targets, mostly membrane proteins, is in close contact with the intra- and extra-cellular matrixes and their three-dimensional structures are inextricably linked to their inclusion in lipid bilayers. Therefore, although binding studies can be performed on the isolated form of these proteins, it remains crucial to measure the affinity of these aptamers in a more physiological environment, i.e., directly on living cells. Here, we describe a procedure for radioactive binding assays that can be adapted for measuring the affinity of aptamers against different cell lines. This method has been semi-automated using a liquid handling robot in order to reproducibly measure the apparent dissociation constant Kd and the apparent number of targets per cell. Relevant issues are discussed including the labeling of aptamers, the cells preparation, the incubation, the washings, the use of non-specific competitors, the data analysis and finally the reporting.

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