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<h2>Protocol</h2> <hr> <ul> <li>Coat 96-w plate with 100 µl/well of 1 µg/ml CD47-Fc (produced in house at Stanford) (in PBS), 4˚C O/N</li> <li>Wash with 1XPBS/0.05% Tween 20 (also called as PBST) </li> <li>Add 100 µl/well of superblock, RT for 1 hr</li> <li>Wash with PBST</li> <li>Add 100 µl/well of 8 serial dilutions of anti-CD47 Ab with starting concentration as 1 µg/ml, and 1:3 dilutions thereafter, RT 1 hr</li> <li>Wash with PBST</li> <li>Add 100 µl/well of HRP conjugated anti mouse Kappa, RT 1 hr</li> <li>Add 100 µl/well of OPT substrate, incubate at room temperature for 5-30 minutes. </li> <li>Add 100 µl/well of 2N H<sub>2</sub>SO<sub>4</sub> to stop the reaction. </li> <li>Read the plate at 490 nm in a plate reader using SOFTmax software.</li> </ul> <hr> <p><br></p> <p>Performed with mouse anti-CD47 antibody, MIAP410 (Clone 3) on plates coated with human CD47-Fc or mouse CD47-Fc. Data available <a href="https://osf.io/z975j/" rel="nofollow">here</a>. <br></p>
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