The following reagents were purchased from Thermo Scientific: BSA (bovine serum albumin in PBS, Imject BSA # 77110), PMPI (p-maleimidophenyl isocyanate, “LINKER” in this study, #28100), Imject™ mcKLH (mariculture keyhole limpet hemocyanin, 5 x 20 mg, in PBS, #77600), and 1-Step™ Ultra TMB (3,3',5,5'-Tetramethylbenzidine, chromogenic ELISA substrate, # 34028). An anti-rabbit horseradish peroxidase-labeled goat IgG was acquired from KPL (#074–1506). Traut’s reagent (2-Iminothiolane HCl) was acquired from ProteoChem (Utah, US, #M3103). POA derivative (5-hydroxyl-2-pyrazinecarboxylic acid, “POA.OH”) was synthesized by Shanghai Haoyuan ChemExpress, Inc. Zeba™ Spin Desalting Columns, 7K MWCO, 2 mL (Thermo, #89889) were used for purification steps. A microplate with hydrophilic/hydrophobic surface (Maxisorp 96-Well Plates, Thermo Scientific, #439454) was employed throughout the study. Young adult rabbits (New Zealand, white females, 2.5–3.0 kg) were acquired from the National Institute of Health (Lima, Peru).
Imject mcklh
Manufactured by Thermo Fisher Scientific
Imject™ mcKLH is a protein derived from the hemocyanin of the keyhole limpet (Megathura crenulata). It is commonly used as a carrier protein in the preparation of immunogenic conjugates for research applications.
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Lab products found in correlation
1 step ultra tmb, by Thermo Fisher Scientific (1 mentions)
Maxisorp 96 well plate, by Thermo Fisher Scientific (1 mentions)
Mabselect sure, by GE Healthcare (1 mentions)
Keyhole limpet hemocyanin (klh), by Enzo Life Sciences (1 mentions)
Imject edc mcklh spin kit, by Thermo Fisher Scientific (1 mentions)
3 protocols using imject mcklh
1
Antibody Production and Purification Protocol
2
CD8+ PBMC Assay with Proprietary Antibodies
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For CD8+ Depleted PBMC assay, the positive assay control (10) (link), keyhole limpet hemocyanin (Imject ™ mcKLH), was purchased from Thermo Fisher Scientific and was reconstituted with 2mL of ultrapure water. The final assay concentration for KLH was 0.33µM. KLH used in DC-T assays with (50ug/ml) and without (25ug/ml) stimulation was purchased from Enzo. Proprietary antibodies, mAb1, mAb2, mAb3, mAb4, and mAb5 were supplied by Eli Lilly and Co. Anti-PCSK9-A, anti-PCSK9-B, anti-IL21R, anti-IL7R, and anti-PDL1 homologs were synthesized using the published sequences described by W.H.O. International Nonproprietary Names for Pharmaceutical Substances or U.S. patents. Whole antibody heavy and light chains were subcloned from the VH and VL genes, respectively. The mAbs of interest were produced by transfection into Chinese Hamster Ovary (CHO-GS/ Lipase KO(2F9) cells (Lonza, Basel, Switzerland)) cells. A Protein-A affinity chromatography (MabSelect SuRe; GE Healthcare Biosciences, AB, Uppsala, Sweden) and Strong Cation Exchange (SCX/SEC) chromatography (Poros50 HS SCX (Thermo Scientific Cat#1335906, GE Healthcare cat#28922937) were used to purify the respective cell culture fluid for each antibody. The final concentration of all the mAbs used in the assay was 50 µg/ml (0.33 µM).
3
Islet Antigen Peptide-KLH Conjugation for T Cell Analysis
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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.
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