Mouse anti his tag monoclonal antibody

Escherichia coli TOP10 and Rosetta (DE3) strains (Invitrogen and Novagen) were cultured in Luria-Bertani broth (LB) with appropriate antibiotics at 37°C. MaxiSorp™ microtiter plates from NUNC were used for ELISA. Rabbit anti-GST IgG antibody conjugated with horseradish peroxidase (HRP) and rabbit anti-Sumo tag IgG antibody were purchased from GenScript (Piscataway, NJ). Mouse anti-His tag monoclonal antibody was obtained from Invitrogen (Waltham, MA). Peroxidase substrate 3,3’,5,5’- Tetramethylbenzidine (TMB) and solution were purchased from Kirkegaard & Perry Laboratories (Gaithersburg, MD). Biacore CM5 chips and amine coupling kit containing 1-ethyl-3-(3 dimethylaminopropyl) carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS) and ethanolamine-HCl were obtained from GE Healthcare (Pittsburgh, PA). Thrombin and FXIII were purchased from Haematologic Technologies, Inc. (Essex Junction, VT). Human plasma Fg was purchased from EMD Millipore (Billerica, MA).

Citrinin (CIT), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS) and 2-(morpholino) ethanesulfonic acid (MES) were purchased from Sigma (St. Louis, MO, USA). TRIZOL and Mouse anti-His tag monoclonal antibody were purchased from Invitrogen (USA). ReverTra Ace-α- was purchased from Toyobo (Japan). The pMD18-T vector and primerSTAR MAX DNA polymerase were purchased from TaKaRa Biotechnology Ltd. (Dalian, China). TNT Quick Coupled Transcription/Translation Systems, Wizard SV Gel and PCR Clean-up System, and RNase-free DNaseІ were purchased from Promega (USA). The pTIG-TRX vector was provided by Beijing Institute of Biotechnology (Beijing, China). A HisTrap HP metal affinity chromatography system was purchased from GE Healthcare (USA). HRP-labeled goat anti-mouse IgG (H+L) was purchased from BioWorld (USA). The CIT-protein conjugate was determined using UV-2550 spectrophotometer (Shimadzu, Japan) and TripleTOF 5600+ (AB SCIEX, USA).

Following SDS-PAGE, proteins separated by molecular weight were electroblotted onto a polyvinylidene difluoride (PVDF) membrane. The membrane was serially probed with mouse anti-His tag monoclonal antibody (1:3000 dilution, Invitrogen, Waltham, MA, USA) and alkaline phosphatase (ALP)-conjugated goat anti-mouse antibody (1:5000 dilution, Invitrogen, Waltham, MA, USA) following blocking with 5% skim milk in phosphate-buffered saline (PBS, 120 mM NaCl, 16 mM Na₂HPO₄, 4 mM NaH₂PO₄, pH 7.4). Finally, color was developed using 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium substrates (G-CLONE, Beijing, China).