transparent plate  (Greiner Bio)

Journal: Scientific Reports

Article Title: Identification of small molecules affecting the interaction between human hemoglobin and Staphylococcus aureus IsdB hemophore

doi: 10.1038/s41598-024-55931-8

Figure Lengend Snippet: Hb binding to IsdB-functionalized plates followed by immunodetection. ( A ) Titration of Y165A IsdB with Hb. 2 pmol StrepTag®II-Y165A IsdB were used to functionalize a Strep-Tactin® XT-coated microplate. Increasing concentrations of Hb in binding buffer were added to distinct wells of the plate and incubated at 4 °C. A 1:1,000 diluted Ab was added to the wells and incubated at 4 °C for 1 h in the dark. Detection was carried out with the TMB substrate incubated for 30 s at 4 °C. The data points represent the average of three replicates. The line through the datapoints is the fitting to Eq. with a K D = 84 ± 11 μM. ( B ) Titration of wt IsdB with Hb. 2 pmol StrepTag®II-IsdB were used to functionalize a Strep-Tactin® XT-coated microplate. Increasing concentrations of Hb in binding buffer were added to distinct wells of the plate and incubated at 4 °C. A 1:1000 diluted Ab was added to the wells and incubated at 4 °C for 1 h in the dark. Detection was carried out with the TMB substrate incubated for 30 min at room temperature. The data points represent the average of three replicates. The line through the datapoints is the fitting to Eq. with a K D = 39 ± 4 nM.

Article Snippet: Absorption spectra of the commercial compounds alone or in the presence of oxyHb were recorded with a microplate reader (TECAN Spark® 10 M) in a 384-well, UV-transparent microplate (Greiner UV-STAR® plate—781801).

Techniques: Binding Assay, Immunodetection, Titration, Incubation