Imject edc mcklh spin kit

We custom-generated rabbit polyclonal antibodies against mouse DGAT1 and DGAT2 (GenScript). To generate DGAT1 antibodies, we used an N-terminal peptide corresponding to amino acids 23–52 (NH2-GGSGPKVEEDEVR DAAVSPDLGAGGDAPAP-COOH) of DGAT1 as the antigen. The peptide was conjugated to mcKLH with the Imject EDC mcKLH spin kit (Thermo Scientific) according to the manual. The antigen was then injected into rabbits to produce the antibody. We injected five rabbits, and two gave a better immune response. To generate DGAT2 antibodies, we used a C-terminal peptide corresponding to amino acids 373–388 (NH2-KTKFGLPETEVLEVN-COOH) of DGAT2 as the antigen. We injected five rabbits, and two gave an appropriate antibody. Antibodies were affinity-purified and used at a 1:1,000 dilution in 5% milk in TBST and incubated overnight at 4°C.

Peptides are conjugated to KLH using an Imject EDC mcKLH spin kit (Thermo Fisher), closely following the manufacturer’s instructions. Briefly, equal amounts of each of four peptides (WE14 or BDC2.5 mimotope, InsB(9–23), GAD65(524–543) (SRLSKVAPVIKARMMEYGTT), and IGRP(206–214) (VYLKTNVFL)) are mixed together and conjugated at a 1:1 ratio (for example 10 mg of peptide mixture with 10 mg of KLH) with mariculture KLH (Imject mcKLH, Thermo Fisher) in Imject EDC conjugation buffer and 15% DMSO. Next, 2.5 mg of the crosslinker EDC (1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride) is added. EDC reacts with exposed carboxyl and amino groups. The reaction is incubated for 2 hours at room temperature. Next, the conjugate is purified by desalting using spin columns. The purified conjugate is sterile filtered and quantified with Nanodrop. Islet Ag-KLH conjugates (10–350 μg) are suspended in PBS and injected i.v. Anti-IL-7Rα antibodies or rat IgG (0.5 mg) are administered i.p. on the same day or 3 days later and repeated on day 4 after the initial antibody administration. PLN and spleen are harvested on day 3 days after the last mAb treatment for analysis of antigen-specific T cells with tetramer enrichment and ELISPOT.