Protein g hrp

Immunoprecipitation complexes obtained from ChIP-TH2B/ ChIP-IgG were eluted in 2X Laemmli buffer and electrophoresed on 15% SDS-PAGE at 100 V for 2.5 h. Proteins were transblotted on Nitrocellulose membrane (Pall bioscience, Pensacola, FL, USA) at 100 V for 1 h 15 min. Non-specific binding to the membrane was blocked with 5% NFDM incubated on a rocker for 1 h at RT. After blocking, the membrane was incubated with anti-TH2B antibody diluted 1:5000 in 1% NFDM and kept overnight at 4 °C with constant rocking. The following day, blots were washed thrice with 0.1% PBST for 5 min and incubated with 1:3000 diluted Protein-G HRP (Thermo fisher scientific, Waltham, MA, USA), which served as secondary antibody, for 45 min with constant rocking. The blots were washed thrice with 0.1%PBST for 5 min and developed using Western Blot Chemiluminescence HRP substrate (TAKARA BIO INC, Otsu, Shiga, Japan).

The commercial IDEXX M. bovis cattle antibody test (#99-29853, Idexx Laboratories, Westbrook, ME, USA), was modified for use in non-bovines by replacing the kit secondary detection anti-bovine-HRP (peroxidase) reagent with either Protein-G-HRP (Sigma-Aldrich, St. Louis, MU, USA #P8170) for deer or Protein-A/G-HRP (Thermo Fisher Scientific, Waltham, MA, USA #32490) for pigs—since Protein-G-HRP did not show good sensitivity for pigs in this test. ELISA plates pre-coated with a cocktail of the immuno-dominant antigens MPB83 and MPB70 were used to test up to 92 serum samples/plates in single wells. Serum samples were diluted 1/50 using the kit sample buffer and 100 μL of diluted sample was loaded onto the ELISA plate and incubated for one hour at room temperature (RT). Plates were washed 6× using kit wash buffer and 100 μL of the secondary detection reagent diluted 1/20,000 in 1%BSA/PBS was added and plates were incubated for 30 min at RT. Plates were washed and developed for 15 min using the kit developing and stop solutions and the Optical Density (OD) was read on an ELISA Reader (range 0–6) at 450 nm (OD450 nm). Kit bovine positive and negative plate controls were included in all tests as test run quality controls (QC). Test readouts are illustrated as the OD450 nm value for each sample (animal).