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 RNAinjected embryos. This represents a straightforward, powerful strategy for confirming antibody specificity in Xenopus. MATERIALS It is essential that you consult the appropriate Material Safety Data Sheets and your institution's Environmental Health and Safety Office for proper handling of equipment and hazardous materials used in this protocol. RECIPES: Please see the end of this protocol for recipes indicated by <R>. Additional recipes can be found online at http://cshprotocols.cshlp.org/site/recipes. Reagents Agarose gel electrophoresis reagents (i.e., 1%-2% agarose gels, loading dye, running buffer, and ethidium bromide) Antibody to test See Protocol: Raising Antibodies for Use in Xenopus (Piccinni and Guille 2020). Cysteine (2% [w/v], pH 7.8; Sigma-Aldrich C7352) Embryo extraction buffer <R> Ficoll (3% [w/v], prepared in 1× MBS; Sigma-Aldrich GE17-0300-50) Freon (1,1,2-trichloro-1,2,2-trifluoroethane; Fisher Scientific T1781) Modified Barth's saline (MBS) (1×, pH 7.8) <R> Nuclease-free dH 2 O (Fisher Scientific 15303711) Reagents for CRISPR analysis only (see Steps 1-9 and Steps 25-33) Cas9 protein (EnGen Cas9 NLS, Streptococcus pyogenes [NEB M0646]) DNeasy Blood & Tissue Kit (QIAGEN) or other appropriate kit to prepare genomic DNA from embryos

The Use of Xenopus for Cell Biology Applications

“…After antigen-specific antibody purification, a robust method is required to determine the specificity and potency of the generated antibody against the antigen in question. In Protocol: Confirming Antibody Specificity in Xenopus, Martin et al (2020) describe a strategy that can be used to verify that the new antibody recognizes its target. This strategy involves knocking down or enhancing the expression of the target in embryos and then performing a western blotting analysis with the new antibody to evaluate its specificity for the target.…”