Confirming Antibody Specificity in Xenopus

Abstract

Verifying that a new antibody recognizes its target can be difficult. In this protocol, expression of a target protein in Xenopus embryos is either knocked down using CRISPR–Cas9 technology (for zygotic proteins) or enhanced by microinjection of a synthetic mRNA (for maternal proteins). Western blotting analysis is then performed. If the antibody recognizes the target protein, the western blot will show a relatively weak band for CRISPR-injected embryos and a relatively strong band for RNA-injected embryos. This represents a straightforward, powerful strategy for confirming antibody specificity in Xenopus.