In the first case, after degranulation, the enzymatic activity of β-hexosaminidase in supernatants and cell pellets solubilized with 1% (v/v) Triton-X-100 in Tyrode's buffer was measured with p-nitrophenyl N-acetyl-β-D-glucosaminide in 0.05 M citric acid (pH 4.5) for 60 min at 37°C.
Because heparin is primarily composed of the disaccharide IdoA 2S -(1 → 4 -GlcNS 6S), the H NMR spectrum of the IS standard in the depolymerization buffer was measured for comparison with that of the heparin depolymerization solution as shown in Fig. 5.
The protein content of samples dissolved in Laemmli buffer was measured using the Pierce 660 nm protein assay (Thermo Scientific, IL, USA).
The serum concentration of hydroperoxides (whole oxidant capacity of serum against N,N-diethylparaphenylene-diamine in acidic buffer) was measured as described previously [ 27].
In comparison, the solubility limit of unmodified glucagon in the same buffer was measured at 0.06 mM (0.2 mg/mL), demonstrating that the XTEN sequence confers an increase in solubility of at least 60-fold.
To examine the isolation efficiency of CFP-10 on films of different nanopore morphology and configuration, the amount of peptide remaining in the wash buffer was measured using indirect ELISA (enzyme-linked immunosorbent assay).
Briefly, 3 μM F-actin (10% pyrenyl labeled) in actin polymerization buffer was measured alone or after the addition of either untagged or GFP-tagged cofilin (1 μM each) at 21°C.
The distribution of [11C]prucalopride between 1-octanol and 0.2 M phosphate buffer (pH 7.4) was measured in triplicate at room temperature by adapting a method previously described [16].
Kidney slides were mechanically lysed in RIPA buffer and fluorescence was measured in a fluorometer (Turner) with excitation of 450 nm and emission of 530 nm.
Cells were then lysed in 50 μl of passive lysis buffer, and luciferase was measured in accordance with the assay method described above.
