His tag mouse monoclonal antibody

CFE reactions were performed to express peptides/enzymes in 1.5 mL microcentrifuge tubes. A standard reaction (15 μL) contained the following components: 12 mM magnesium glutamate, 10 mM ammonium glutamate, 130 mM potassium glutamate, 1.2 mM ATP, 0.85 mM each of GTP, UTP, and CTP, 34 μg/mL folinic acid, 170 μg/mL of E. coli tRNA mixture, 2 mM each of 20 standard amino acids, 0.33 mM nicotinamide adenine dinucleotide (NAD), 0.27 mM coenzyme A (CoA), 1.5 mM spermidine, 1 mM putrescine, 4 mM sodium oxalate, 33 mM phosphoenolpyruvate (PEP), 5-15 nM gene template (plasmid or linear DNA), and 27% (v/v) of cell extract. Where applicable, a protein inhibitor cocktail was added to the reaction (1x final concentration) to prevent the degradation of unmodified precursors. All reactions were incubated at 30 °C for 6 h before further analysis of the synthesized peptides and enzymes. If required, the reaction volume of CFE can be scaled up accordingly. For Western-blot analysis, His-tag labeled peptides/enzymes were visualized by using the primary antibody His-Tag Mouse Monoclonal Antibody (catalog number: 66005-1-Ig, 1:10000 dilution, Proteintech) and the secondary antibody HRP-Conjugated Affinipure Goat Anti-Mouse IgG(H + L) (catalog number: SA00001-1, 1:10000 dilution, Proteintech).

The recombinant protein of EmROM5 was analyzed by Western blot assays. After the SDS-PAGE of rEmROM5, it was transferred to nitrocellulose membranes (Merck Millipore, Tullagreen, Carrigtwohill, Ireland). Then, the membranes were incubated in the primary antibody of chicken anti-E. maxima serum (1:50 dilution) or His-Tag Mouse Monoclonal antibody (1:8000 dilution, Proteintech, Wuhan, China)at ambient temperature for 4 h separately, and incubated in the secondary antibody of horseradish peroxidase (HRP)-conjugated goat anti-chicken IgG (1:4000 dilution, Biodragon-immunotech, Beijing, China) or HRP-conjugated anti-mouse (1:10,000 dilution, Thermo Fisher Scientific, Waltham, MA, USA) at 37 °C for 1.5 h separately. Meanwhile, uninfected chicken serum was set for negative control as a primary antibody. Finally, an Enhanced HRP-DAB substrate chromogenic kit (TIANGEN Biotech, Beijing, China) or ECL chemiluminescence detection kit (Vazyme, Nanjing, China) was used for color rendering.

The prokaryotic expression strain E. coli Rossetta (DE3) was transformed with the correctly sequenced plasmid. For prokaryotic expression analysis, the strains were cultured on a rotary shaker with incubator at 37 °C and 200 rpm speed. During log phase, an isopropyl β-d-thiogalactopyranoside (IPTG) concentration of 0.4 mM was used to induce the fusion protein expression at 37 °C for 6 h. The bacterial suspension was harvested by centrifugation for 0, 2, 4, and 6 h, and the fusion protein was separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and visualized using Coomassie brilliant blue (CBB) staining. Protein samples were transferred to an Immobilon Polyvinylidene Fluoride (PVDF) membrane (Millipore, Burlington, MA, USA), 5% skim milk powder was used to block the membrane. Membranes were treated with His-Tag mouse monoclonal antibody (Proteintech) and a secondary antibody of horseradish peroxidase (HRP) conjugated affinipure goat anti-mouse IgG (H + L) (Proteintech), and detected using Chemiluminescence Ecl Detection Kit (Millipore, Burlington, MA, USA).