Elisa pod substrate tmb kit

The standard sample of Sav (Nacalai Tesque, Kyoto, Japan) and Sav^nat produced by S. lividans were immobilized on biotin coated plates. The maximal amount of each Sav added to the plates was 300 μg/mL. Each Sav was incubated in a 96-well biotin-coated plate (Thermo Fisher Scientific) for 30 min at 4°C. The wells were then washed with TBST three times, and 5 μg/L biotinylated horseradish peroxidase (biotin-HRP) (Thermo Fisher Scientific) per well were incubated for 1 h at room temperature. After washing wells with TBST three times, HRP activity was assayed with an ELISA POD substrate TMB Kit (Nacalai Tesque). Then the absorbance of 450 nm was analyzed with a plate reader (Wallac 1420 ARVOsx).
Sav^Eco, Sav^ΔN, and Sav^ΔC were immobilized on nickel-coated plates (Thermo Fisher Scientific). The maximal amount of each Sav added to the plate was 300 μg/mL. After immobilization of each Sav, HRP activity was assayed in a similar fashion.

Total protein concentrations of lysates were measured using a Pierce BCA protein assay kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. Equal amounts of protein were used for enzyme-linked immunosorbent assay (ELISA). The lysates (0.5 mg protein) were coated onto 96-well microplates for 20 h at 4°C. After washing the plates three times by filling the wells with 200 μL phosphate-buffered saline (PBS), the remaining protein-binding sites in the coated wells were blocked by adding 200 μL of 5% FBS (Merck, Darmstadt, Germany) in PBS. After the plates were washed three times, 100 μL of primary anti-NAT2 monoclonal antibody (1:100; cat. no. sc-134399; Santa Cruz Biotechnologies, Dallas, TX, United States) was added to each well, and plates were then incubated for 1 h. After washing three times, 100 μL of HRP-Rabbit Anti-Mouse IgG (H + L) Conjugate (1:100,000; Thermo Fisher Scientific) was added to each well, and plates were incubated for 30 min. After washing three times, we added 100 μL of an ELISA POD substrate TMB kit (Nacalai Tesque, Kyoto, Japan) to each well and incubated the plates for 10 min. An equal volume of 1 M HCl as stopping solution was added, and the optical density at 450 nm was measured using a microplate reader (ARVOmx; PerkinElmer, Waltham, MA, United States).

The mCCR9 peptides, such as wild type (WT), 19 of 1× alanine (1× Ala)-substituted peptides (Table 1), and 18 of 2× alanine (2× Ala)-substituted peptides (Table 2), were synthesized using PEPscreen (Sigma-Aldrich Corp., St. Louis, MO, USA). Each peptide was immobilized on Nunc Maxisorp 96-well immuno plates (Thermo Fisher Scientific, Inc.) at a concentration of 1 μg/mL for 30 min at 37 °C. As a negative control, no peptide was immobilized on the immuno plates. After washing with phosphate-buffered saline containing 0.05% Tween20 (PBST), the wells were blocked with 1% bovine serum albumin containing PBST for 30 min at 37 °C. The plates were then incubated with C₉Mab-24 (1 μg/mL), followed by a 1:20,000 dilution of peroxidase-conjugated anti-rat immunoglobulins (Sigma-Aldrich Corp.). Enzymatic reactions were performed using an ELISA POD Substrate TMB Kit (Nacalai Tesque, Inc.). Optical density was detected at 655 nm using an iMark microplate reader (Bio-Rad Laboratories, Inc., Berkeley, CA, USA).