Horseradish peroxidase hrp conjugated anti goat antibody

N-terminal biotinylated peptides of amino acids 384 to 411 (the 27 amino acids of the HVR1) of genotype 1a H77, genotype 2a J6, genotype 2a JFH-1, and amino acids 412 to 443 (the amino acids directly downstream of HVR1) of J6 and JFH-1 as well as the scrambled control of the 2a JFH-1 HVR1 peptide were synthesized by GL Biochem. Peptides were added to neutravidin-coated 96-well plates at 0.5 μg/well for 1 hour. Plates were blocked with 5% bovine serum albumin (BSA). The presera or postvaccination polyclonal E1/E2 antisera from goat 757 were added to wells in 3-fold serial dilutions starting with 1/50. Horseradish peroxidase (HRP)-conjugated anti-goat antibody (Santa Cruz Biotechnology) was used to detect goat antisera binding to peptides. Plates were developed using tetramethylbenzidine (TMB) substrate (Mandel Scientific), and the reaction was stopped after 4.5 minutes. The EnSpire 2300 multilabel reader (Perkin-Elmer) was used to record absorbance values for an optical density (OD) of 450 nm. Nonspecific absorbance values for negative controls were subtracted from experimental wells, and data were plotted using GraphPad Prism 7 software.

Alanine transaminase (ALT) and aspartate transaminase (AST) color endpoint assay kits were purchased from Bio Scientific (Austin, TX). BM chemiluminescence blotting substrate (POD) and 10X blocking reagent were obtained from Roche Diagnostics, Inc. (Indianapolis, IN). Celastrol was purchased from BOC Sciences (Shirley, NY). iScript cDNA synthesis kit and SYBR Green super mix were bought from Bio-Rad (Hercules, CA). Horseradish peroxidase (HRP)-conjugated anti-goat antibody was obtained from Santa Cruz Biotechnologies (Santa Cruz, CA). GAPDH-specific antibody (#2118) and Horseradish peroxidase (HRP)-conjugated anti-rabbit antibodies were purchased from Cell Signaling Technology (Beverley, MA). Lipocalin-2/NGAL antibody (AF1857), its corresponding ELISA kit (MLCN20), and recombinant mouse leptin protein were obtained from R&D Systems (Minneapolis, MN). Mouse leptin and ultra-sensitive mouse insulin ELISA kits were purchased from Crystal Chem, Inc. (Downers Grove, IL). TRIzol reagent was obtained from Invitrogen (Carlsbad, CA). RNeasy MinElute Cleanup Kit was bought from Qiagen (#74101, Valencia, CA).