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Lyophilized samples were resuspended in assay buffer without BSA.
Streptavidin-coated donor beads were prepared in assay buffer at a concentration of 100 μg/mL.
Alterations in assay conditions can restore quantitation of this subtype.
Bound proteins were eluted in 6 × 25 µl steps in assay buffer supplemented with 2.5 mM d-desthiobiotin.
The cGMP standard was prepared as a serial dilution from 0.5 nM to 5 μM in assay buffer without BSA.
Briefly, synaptosomes were suspended in assay buffer containing 15 mM p-NPP and incubated at 37 °C for 30 min.
Cells were equilibrated in assay plates via centrifugation and placed in incubators for 24 h before treatment.
Sequential improvement in assay performance included anti-human immunoglobulin-enhanced, complement-dependent cytotoxicity techniques followed by cell-based flow cytometry.
DHF and NADPH stocks were made up to 1 mM in assay buffer (0.1 M Tris, pH 7.4) and stored at −20 °C until use.
The following day, cultures were incubated in assay medium with 1 nM or without insulin (PAN-Biotech) for 30 minutes at 37 °C.
Peptide inter-run variability is important to consider in assay development because a target with high inter-run variability would lead to low confidence peptide quantifications.
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