Raising Antibodies for Use in Xenopus

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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 . 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 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) 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

Section: Discussionmentioning

confidence: 99%

Confirming Antibody Specificity in Xenopus

Martin¹,

Page

Piccinni³

et al. 2020

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Assessing the Immune Response When Raising Antibodies for Use in Xenopus

Piccinni

Guille

2020

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Purifying Antibodies Raised against Xenopus Peptides

Piccinni

Guille

2020

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Antibody production for work in Xenopus involves the immunization of a host with an antigen, usually a Xenopus protein or peptide alien to the host. The antibody-containing serum, normally returned to the investigator by the company/bioresource unit where it was raised, is comprised of all proteins not used in blood clotting (coagulation) and all the electrolytes, antibodies, antigens, hormones, and any exogenous substances, such as drugs and microorganisms, that were in the blood. It is often necessary to separate the target antibody from the rest of the serum components to minimize nonspecific protein–antibody interactions in downstream applications (e.g., when performing western blotting). Most antibody production companies provide a column containing the peptide coupled to glass beads. A purification procedure for using this type of column (i.e., one that is based on controlled-pore glass beads) is described here.