I want to inhibit clathrin-mediated endocytosis of antibody-targeted receptor using chlorpromazine-HCl as inhibitor.
The hypothesis is that chlorpromazine will inhibit clathrin-dependent endocytosis, and the therapeutic antibody-receptor complex should retain on cell surface after treatment.
The question is should I treat the cells with antibody+ chlorpromazine HCl/medium or pre-block the cells with chlorpromazine HCl/medium follow by replacing with antibody-only treatment medium?
Malcolm: As chlorpromazine-HCl disrupts the acidic pH of endosomes thus blocking its formation/movement, you would pretreat the cells at least one hour prior to the antibody incubation to ensure plasma surface-only binding. I'd simply add chlorpromazine-HCl and incubate for 1 hr, followed by adding an aliquot of concentrated antibody solution and incubate for additional time period. Good luck.
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