Assessing the Immune Response When Raising Antibodies for Use in Xenopus

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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.

mentioning

confidence: 99%

Purifying Antibodies Raised against Xenopus Peptides

Piccinni

Guille

2020

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Raising Antibodies for Use in Xenopus

Piccinni

Guille

2020

Self Cite

For work in Xenopus, frog-specific antibodies must usually be raised, although a few antibodies against mammalian proteins cross-react. To produce an immunogen for antibody production, human embryonic kidney (HEK) expression systems can be used as described here. For most laboratories, the actual method of raising the antibody is determined by local ethical regulations controlling the adjuvant and injection protocols used. Because these steps are often outsourced, they are not included in this protocol.

The Use of Xenopus for Cell Biology Applications

Philpott

2021

Problems of cell biology and the molecular controls underpinning them have been studied in the remarkably versatile Xenopus systems for many years. This versatility is showcased in several accompanying protocols, which are introduced here. One protocol demonstrates how the Xenopus embryonic ectoderm can be used to study the effects of mechanical cell deformation; another illustrates how the developing eye can be used as a platform for determining cell-cycle length. Two protocols show how extracts from Xenopus embryos can be exploited to characterize the behavior of specific intracellular proteins-specifically, to determine protein phosphorylation status and the ability to bind to chromatin. Finally, because specific antibodies to Xenopus proteins are pivotal reagents for cell biology and biochemistry applications, four protocols describing how to generate, purify, and assay the specificity of antibodies raised against Xenopus proteins are included in hopes of stimulating the expansion of these critical resources across the Xenopus community.