68Ga-DOTATATE synthesis: Using method (i) the precursor (25μg) in 1.5 mL 0.25M Na-acetate buffer was reacted with the 6 mL eluate (pH reaction 3.5-4) for 7 min at 90-95°C 90-95°C
Ubiquitin in neutral PBS buffer was reacted with DSP for cross-linking, followed with trypsin digestion and EC/DESI-MS analysis.
After removing the activated charcoal with a 0.2 μm filter, an aliquot of extraction buffer was reacted with anthrone-sulfuric acid at 95°C for 15 min and then cooled to room temperature.
Following FGE treatment, Fcs were buffered exchanged into labeling buffer and were reacted with AF488 hydroxylamine as stated above.
After blocking with the blocking buffer, cells were reacted with the anti- C. paradoxa DipM antibodies (1 100 in the blocking buffer) for 4 h at room temperature.
After the membranes were washed with TBST buffer, they were reacted with the appropriate horseradish peroxidase-conjugated secondary antibodies for 30 min at room temperature.
After washing in PBS and Tris-buffer, they were reacted with a solution containing 0.01% diaminobenzidine hydrochloride, 0.025% nickel ammonium sulfate, 0.025% cobalt chloride and 0.035% hydrogen peroxidase in Tris-buffer.
Ferric monomeric WT, H81V and H81Y Cygb (50 μM), in 20 mM phosphate buffer (pH 7.4), were reacted with a range of hydrogen peroxide (Sigma, UK) concentrations (0 5 mM) and incubated at room temperature for 18 hours.
Monomeric WT, H81V and H81Y Cygb (5 μM) in 0.1 M sodium phosphate buffer pH 7.4 were reacted with hydrogen peroxide (5 mM) in the presence of 8.94 mM guaiacol (0.1% v/v, Sigma, UK).
Monomeric WT, H81V and H81Y Cygb (80 μM) in 20 mM sodium phosphate buffer (pH 7.4) were reacted with hydrogen peroxide, either at 1 1 at (80 μM) or 1 10 (800 μM) protein:hydrogen peroxide ratio in Wilmad SQ EPR tubes (Wilmad Glass).
After washing with TBS buffer, the primary immune complexes were reacted with biotinylated secondary antibodies provided in ABC kits (Vector Laboratories, Inc., Burlingame, CA, USA) and the resulting complexes were detected upon the addition of a chromogen, 3-amino-9-ethylcarbazole (AEC, DakoCytomation, Inc., Carpinteria, CA, USA).
