Protocol for screening and validating antibodies specific to protein phosphorylation sites using a set of yeast biopanning approaches

Monika Arbaciauskaite; Azady Pirhanov; Justin Paoloni; Yu Lei; Yong Ku Cho

doi:10.1016/j.xpro.2024.103241

Protocol for screening and validating antibodies specific to protein phosphorylation sites using a set of yeast biopanning approaches

STAR Protoc. 2024 Sep 20;5(3):103241. doi: 10.1016/j.xpro.2024.103241. Epub 2024 Aug 1.

Authors

Monika Arbaciauskaite¹, Azady Pirhanov², Justin Paoloni³, Yu Lei⁴, Yong Ku Cho⁵

Affiliations

¹ Department of Chemical and Biomolecular Engineering, University of Connecticut, Storrs, CT 06269, USA. Electronic address: [email protected].
² Department of Biomedical Engineering, University of Connecticut, Storrs, CT 06269, USA.
³ Department of Chemical and Biomolecular Engineering, University of Connecticut, Storrs, CT 06269, USA.
⁴ Department of Chemical and Biomolecular Engineering, University of Connecticut, Storrs, CT 06269, USA; Department of Biomedical Engineering, University of Connecticut, Storrs, CT 06269, USA.
⁵ Department of Chemical and Biomolecular Engineering, University of Connecticut, Storrs, CT 06269, USA; Department of Biomedical Engineering, University of Connecticut, Storrs, CT 06269, USA; Institute for Systems Genomics, University of Connecticut, Storrs, CT 06269, USA. Electronic address: [email protected].

Abstract

Developing antibodies with high specificity against post-translationally modified epitopes remains a challenge. Yeast biopanning is well suited in screening for high-specificity binders. Here, we present a protocol for screening and validating antibodies specific to protein phosphorylation sites using a set of yeast biopanning approaches. We describe steps for screening a yeast surface display library for antibodies and other binders. We then detail procedures for validating the antibodies found by analyzing their specificity through whole-well image analysis in 96-well plates. For complete details on the use and execution of this protocol, please refer to Arbaciauskaite et al.¹.

Keywords: Antibody; Biotechnology and bioengineering; High-throughput Screening.

MeSH terms

Antibodies / immunology
Antibodies / metabolism
Epitopes / immunology
Epitopes / metabolism
Peptide Library
Phosphorylation
Protein Processing, Post-Translational
Saccharomyces cerevisiae* / metabolism

Substances

Antibodies
Epitopes
Peptide Library

Grants and funding

R01 AG083876/AG/NIA NIH HHS/United States