5 µl aliquots of the PCR products with 1 µl loading buffer were analyzed by electrophoresis using a 1% (w/v) agarose gel in Tris Boric acid EDTA (TBE. 1X) buffer at 100 V for 45 min.
With the aid of GIS software ArcView 3.2 and landscape pattern analysis software FRAGSTATS 3.3, the landscape spatial patterns of each buffer zone were analyzed at the landscape level and class level.
After washing in FACS buffer, the cells were analyzed at λex 552/ λem 580 nm.
The sample stored at −80 °C in pH 7.4 buffer was analyzed by LC/MS/MS to determine whether sites in addition to Tyr 411 were labeled.
TE buffer was analyzed as blank.
Samples were analyzed at a concentration of 2 mg/ml in 20 mM His pH 6.5 buffer and at 1.0°C/min scan rate.
All HDX experiments were carried out at 4 °C in 80% in D2O (20 mM Tris (in D2O, pD 7.6), 0.5 M NaCl, 0.5 mM TCEP) where 4 μL of protein sample (14 μM PAD2 in 20 mM Tris pH 7.6, 0.5 M NaCl, 0.5 mM TCEP, 10% glycerol) that was incubated for 60 min with either 0.5 mM EDTA or 2.5 mM CaCl2 was mixed with 16 μL D2O buffer, and HDX was analyzed at eight time points (0, 0.17, 0.5, 1, 2, 15, 60, 240 min).
The proteins were eluted in 2 ml fractions using same buffer and each fraction was analyzed at 280 nm to detect the presence of proteins.
Temperature melt experiments were conducted at the same concentrations and buffer conditions, except for Stx2B-Q40L, wasch wanalyzedzed at 55 µM protein concentration in 10 mM phosphate buffer, 150 mM NaCl.
Beads were then washed with NETN buffer three times, and samples were boiled with 2× SDS loading buffer, and were analyzed by immunoblot with indicated antibodies.
