Nhs lc biotin reagent

Antibody PSR binding was carried out following previously described methods [44 (link),45 (link),46 (link)]. In summary, CHO cells were used to extract soluble membrane protein (SMP) and soluble cytosolic protein (SCP) fractions, which were subsequently biotinylated using the NHS-LC-Biotin reagent from Thermo Fisher Scientific (Waltham, MA, USA) (#A39257). Yeast-presented IgGs were then incubated with a 1:10 dilution of the biotinylated SMP and SCP stocks for 20 min at ice-cold temperatures, washed twice with PBSF [47 (link)], and stained with a secondary labeling mix composed of ExtrAvidin-R-PE from Sigma-Aldrich (St. Louis, MO, USA) (#E4011), anti-human LC-FITC from Southern Biotech (Birmingham, AL, USA) (#2062-02), and propidium iodide from Sigma-Aldrich (#11348639001) for 15 min on ice. The cells were subsequently washed with PBSF and suspended in PBSF for flow cytometric analysis on a BD FACS Canto II instrument from BD Biosciences (San Jose, CA, USA). The binding mean fluorescence intensity (MFI) was determined with a flow cytometry analyzer and normalized to a score ranging from 0 to 1 using three control antibodies that indicate low, medium, and high binding to the PSR reagent.

N-terminal amidated peptides were synthesized by GL Biochem, Ltd. (Shanghai, China). Each peptide was individually conjugated with BSA using Sulfo-SMCC (Thermo Fisher Scientific, MA, USA) according to the manufacturer’s instructions. Briefly, BSA was activated by Sulfo-SMCC at a molar ratio of 1:30, followed by dialysis in PBS buffer. The peptide with cysteine was added at a w/w ratio of 1:1 and incubated for 2 h, followed by dialysis in PBS to remove the free peptides. A few conjugates were randomly selected for examination by SDS-PAGE. For the biotin-BSA-peptide conjugates, before conjugation, BSA was labeled with biotin by using NHS-LC-Biotin reagent (Thermo Fisher Scientific, MA, USA) at a molar ratio of 1:5 and then activated by Sulfo-SMCC.