CORE B – PROTEINS & ASSAYS

CORE B – PROTEINS & ASSAYS ABSTRACT The APOBEC3 (A3) family of single-stranded DNA-cytosine deaminases function normally to defend cells from invading viruses. Overwhelming evidence implicates at least two of these family members, APOBEC3A (A3A) and APOBEC3B (A3B), in causing mutagenesis in a wide variety of primary cancers, with metastases frequently showing increased mutation burdens attributable to these enzymes. Over the previous funding period, this Core successfully developed protein expression methods capable of producing up to milligram quantities of highly active A3 enzymes and identified custom antibodies (in-house and in collaboration with industry) that are specific to both A3A and A3B and useful for a wide variety of assays including immunoblot, IHC, immunofluorescent imaging, and ELISA. In addition, this Core has developed novel assays for determining the activity of A3 enzymes and has performed these assays to support the scientific activities of the Program. Here, Core B – Proteins & Assays will continue to support the Program through two specific aims. Aim 1 will continue to produce essential proteins including A3 enzymes and anti-A3 monoclonal antibodies for use by Program members, Program collaborators, and the broader scientific community. Aim 2 will continue to perform standardized activity and binding assays for Program members and collaborators, and additionally, develop new assays and standard operating procedures (SOPs) for use by Program members and the broader scientific community to ensure inter-lab reproducibility. The availability of these standardized reagents and assays will enable the Projects and Cores to continue to advance the overall goal of blocking A3A/B-dependent tumor evolution. Rigor and reproducibility are enhanced by having a single Core produce standardized reagents and perform key assays for each of the Projects and other Cores, while additionally reducing the overall cost by centralizing these essential services.