Monoclonal anti polyhistidine peroxidase

Cells were lysed by sonication in Tris‐buffered saline (TBS) buffer supplemented with 1 mM phenylmethylsulfonyl fluoride (PMSF) and 1 μl/ml of protease inhibitor cocktail (Set IV, EMD Chemicals, Inc). Protein pull‐down experiments were performed using FLAG Beads (L00432‐10, A2S) according to the manufacturers' recommendations. Proteins were eluted using free FLAG‐peptides (F3290, Sigma). Western blot analysis was performed using THE™ DYKDDDDK Tag Antibody [HRP‐conjugated] (A01428, GenScript) and Monoclonal Anti‐polyHistidine−Peroxidase (A7058, Sigma). Proteins were analyzed on 4–20% SurePAGE precast gels (M00657, GeneScript).

For preparation of extracts by alkaline lysis, overnight cell cultures were centrifuged and pellets resuspended in 0.2 M NaOH and 0.2% β-mercaptoethanol then left on ice for 10 min. Soluble protein was precipitated by addition of 5% TCA for a further 10 min on ice. After centrifugation (13,000 g for 5 min at 4°C), soluble protein was resuspended in 10 µl of 1 M Tris-HCl, pH 9.4, and boiled in 90 µl of SDS-PAGE sample loading buffer for 10 min. Samples (1–2 OD₆₀₀ equivalent) were resolved by SDS-PAGE followed by immunoblotting. Monoclonal anti-GFP antibody was obtained from Roche (11814460001). PGK1 was used as a control (A6457; Invitrogen). Secondary antibody was HRP-linked anti–mouse polyclonal (1706516; Bio-Rad Laboratories). Monoclonal anti-polyhistidine−peroxidase was obtained from Sigma-Aldrich (A7058-1VL).
All blots were blocked in 2% (wt/vol) fat-free Marvel milk in TBS–Tween 20 (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 0.1% [vol/vol] Tween 20). Tagged proteins were detected and imaged by chemiluminescence imaging.

Colonies containing the synthetic fragment of 603 bp (carrying PelB, N-SH2 domain, enterokinase cleavage site sequence and LTS) were inoculated into 20 ml of LB broth having 0.1 mg/ml ampicillin and grown overnight at 37°C. These cultures were used to inoculate 1000 ml of LB broth encompassing 0.1 mg/ml ampicillin and grown to an OD₆₀₀ of 1. This was preceded by induction with the final concentration of 0.5 mM IPTG (Cinnagen Company, Iran) at 30°C overnight. The following day, the cultures were centrifuged at 8500 × g for 15 min and the supernatant was poured away.
Subsequently, the expression of the fusion protein was monitored by SDS-PAGE analysis (12% polyacrylamide, Sigma) [36 (link)]. Western blotting was used to analyze the expression of the fusion protein. The membrane was incubated with a 1:1000 dilution of monoclonal anti-polyhistidine-peroxidase (Sigma, USA). Proteins were visualized using H₂O₂/DAB substrate/ chromogen (Sigma, USA).