However, we found out that the sequencing outcomes were less satisfactory and counter-productive due to ambiguous base calls in our cases. Purification of antibody Timing: 1?week 8. Cell culture supernatants of the hybridomas generated as part of (Ahn et?al., DKFZp781H0392 2021; Nguyen et?al., 2021) were prepared by centrifugation for 15?min at approximately 6,000? at 4C using JLA8.1 rotor, followed by filtration through 0.2?m cellulose acetate filters. 9. Protein G resins were packed in a 25?mL gravity plastic column and equilibrated by flowing 20?mL of the binding buffer (20?mM sodium phosphate buffer, pH 7.0). 10. The cleared hybridoma supernatants from step 8 were submitted to the prepared Protein G resin from step 9 three times. 11. The antibody-bound resins were washed with 25C50?mL of the binding buffer. 12. The bound antibodies were eluted by applying 10?mL of the elution buffer (0.1?M glycine-HCl, pH 2.7), which was harvested in a conical tube containing 1?mL of the equilibration buffer (1?M Tris-HCl, pH 8.0) for immediate neutralization. 13. The purity and yield of purified antibodies were monitored via 15% SDS-PAGE. 14. Purified antibodies were divided in 50?L aliquots and stored at ?80C until use. Flash freezing with liquid nitrogen was not required in this case. determining antibody recognition sites on various antigens. For complete details on the use and execution of this protocol, please refer to Ahn et?al. (2021) and Nguyen et?al. (2021). Subjet areas: Immunology, Microbiology, Microscopy, Antibody, Protein Biochemistry, Structural Biology, Cryo-EM Graphical abstract Open in a separate window Highlights ? This protocol describes the entire procedures for determining antibody target sites ? One bacterial AB toxin and toxin-recognizing mAb pair was used as an example ? IgG can be directly used to determine the B cell epitope map ? This protocol offers MRT68921 dihydrochloride insights into other projects concerning mAb-antigen complexes This cryo-EM protocol was used to determine the B cell epitope map on the CdtB subunit of typhoid toxin, an A2B5 toxin secreted by Typhi during infection. Immunoglobulin G (IgG) was directly mixed with typhoid toxin in this protocol, different from our previous cryo-EM protocol that uses the Fab fragments in place of IgG. This simple approach requires smaller amounts of materials, supporting the broader use of this protocol for determining antibody recognition sites on various antigens. Before you begin This protocol describes specific steps for determining the B cell epitope map of IgG targeting the CdtB subunit of typhoid toxin. Buffer preparation Timing: 1?day Antibody purification 1. Binding buffer C Store at 4C for up to 1?month Prepare 1?L of the Protein G binding buffer containing 20?mM sodium phosphate, pH 7.0. 2. Elution buffer C Store at 4C for up to 1?month Prepare 100?mL of the elution buffer containing 0.1?M glycine-HCl, pH 2.7. 3. Equilibration buffer C Store at 4C for up to 1?month Prepare 100?mL of the equilibration buffer containing 1?M Tris-HCl, pH 8.0. Antigen purification 4. Buffer A C Store at MRT68921 dihydrochloride 4C for up to 1?month Prepare 200?mL buffer A containing 15?mM Tris-HCl, pH 8.0, 150?mM NaCl, 20?mM imidazole. Buffer A DH5, a single colony was isolated, and the isolated plasmid was Sanger-sequenced using either T7 promoter or T7 terminator primer (Table 1). Other plasmids and strains can be used. CRITICAL: The PCR amplicons could be directly sequenced to determine the antibody variable regions MRT68921 dihydrochloride via Sanger sequencing. However, we found out that the sequencing outcomes were less satisfactory and counter-productive due to ambiguous base calls in our cases. Purification of antibody Timing: MRT68921 dihydrochloride 1?week 8. Cell culture supernatants of the hybridomas generated as part of (Ahn et?al., 2021; Nguyen et?al., 2021) were prepared by centrifugation for 15?min at approximately 6,000? at 4C using JLA8.1 rotor, followed by filtration through 0.2?m cellulose acetate filters. 9. Protein G resins were packed in a 25?mL gravity plastic column and equilibrated by flowing 20?mL of the binding buffer (20?mM sodium phosphate buffer, pH 7.0). 10. The cleared hybridoma supernatants from step 8 were submitted to the prepared Protein G resin from step 9 three times. 11. The antibody-bound resins were washed with 25C50?mL of the binding buffer. 12. The bound antibodies were eluted by applying 10?mL of the elution buffer (0.1?M glycine-HCl, pH 2.7), which was harvested in a conical tube containing 1?mL of the equilibration buffer (1?M Tris-HCl, pH 8.0) for immediate neutralization. 13. The purity and yield of purified antibodies were monitored via 15% SDS-PAGE. 14. Purified antibodies were divided in 50?L aliquots and stored at ?80C until use. Flash freezing with liquid nitrogen was not required in this case. Purified antibodies were stored stably without excipients such as glycerol. However, if desired, flash freezing and the addition of excipients like glycerol can be considered in this step. IgG was directly used for the complex structure determination for TyTx11 mAb (Ahn et?al., 2021). However, if desired, the following steps can be carried out to prepare the Fab fragment, as described in (Nguyen et?al., 2021). 15. Fab fragment generation:a. Concentrate purified mAbs to 20?mg/mL in.