Figure 3.

OX133 binds to reduced and labeled proteins at the cell surface and after immobilisation to support surfaces. 2B4 cells (1 × 10⁶) were treated for 30 minutes with TCEP (2.5 mM), TRX (1.25 ug) or PBS as non-reduced (NR) control (both panels). Washed cells were alkylated with NEM (5 mM) and the labile disulfides revealed were labeled with OX133 (1:100) for 20 minutes. Binding was detected by an anti-mouse FITC-conjugate (1:200) followed by flow analysis on a FACSCalibur flow cytometer (BD Biosciences). 10,000 gated (live cell) events were recorded in 4 separate experiments: NR (light gray histogram); TCEP (panel (A)) or TRX (panel (B)) reduction (dark gray histogram). Median fluorescence values for all data revealed that TCEP reduction enhanced NEM labeling of labile disulfides most strongly (*p < 0.05) (C). 2B4 cell lysates from TCEP reduced and NEM alkylated samples were precipitated using sepharose-coupled OX133, separated by SDS-PAGE and Western blotted using OX133 antibody (1:1000) and an anti-mouse conjugate (D).