Anti his hrp antibody

Immulon 2-HB 96-well plates (VWR) were coated overnight at 4°C with 50 μl of partially purified mucins from bovine submaxillary glands (type I-S; Sigma) and porcine stomachs (type II and III; Sigma) at 100 μg/ml in 50 mM Carbonate/Bicarbonate pH 9.6. Wells were blocked for 1 hr at 25°C with 200 μl of 0.1% (w/v) bovine serum albumin (BSA) in PBS–0.05% Tween 20 and then washed once with 200 μl of incubation buffer (0.05% (w/v) BSA in PBS–0.05% Tween 20). Wells were then incubated for 3 hrs at 25°C with 50 μl of ChiA-FL, ChiA-NT, ChiA-N1, ChiA-N2, ChiA-N3, ChiA-CTD, ChiA-CTD^D504A, ChiA-CTD^H506A, ChiA-CTD^E543M, ChiA-CTD^H544A, ChiA-CTD^N547A, ChiA-CTD^Q583A, ChiA-CTD^Q595A, ChiA-CTD^Q617A, NttE and SslE at 10 μM in incubation buffer. This was followed by four washes with 200 μl of incubation buffer and incubation with 50 μl of anti-His-HRP antibody (Sigma), diluted 1:2000 in incubation buffer for 1 hr at 24°C. After four washes with 200 μl of incubation buffer, 150 μl of o-Phenylenediamine dihydrochloride (Sigma) was added for 30 min and then data was recorded at 450 nm.

Trypticase peptone yeast extract glucose (TPYG) and Luria Bertani (LB) medium were purchased from Difco Laboratories, USA. Escherichia coli SG13009, pQE30-UA vector, Ni–NTA resin, plasmid DNA purification kit (27,106), and PCR Kit were procured from Qiagen, USA. Pre-stained protein markers, unstained protein molecular weight marker, (SM0431), DNA ladder (SM0313) were obtained from Fermentas, USA. Primers were synthesized from Microsynth, Switzerland. Cell culture media components, monoclonal anti-polyhistidine antibody raised in mouse (H1029), HRP conjugate AP163P, Anti-His HRP antibody, mouse monoclonal antibody isotyping reagents (ISO2) and chicken anti-goat Ig antibody, 3, 3′-diaminobenzidine (DAB), and other required chemicals were purchased from Sigma Aldrich, USA. Cytokine ELISA kit & flowcytometry reagents were obtained from BD Biosciences, USA.

Lipid strips (Echelon Biosciences) were blocked at room temperature for 1 h in TBST (50 mM Tris pH 7.5, 3 mM KCl, 137 mM NaCl, 0.1% Tween 20) containing 3% BSA and then incubated overnight with 300 pmol His-tagged NttA. The membrane was washed three times with blocking buffer and then incubated for 2 h at room temperature in the same buffer containing anti-His-HRP antibody (Sigma) diluted 1:2,000 and then treated with enhanced chemiluminescence substrate (ECL; Pierce) before detection by enhanced chemiluminescence.