Hplc grade isopropanol

Frozen Antarctic krill with a water content of 77.8 ± 2.8% (w/w) was provided by Liaoning Province Dalian Ocean Fishery Group Co., Ltd. (Dalian, China) and stored at −70 °C. PC, PE, EPA, DHA and astaxanthin standards, 2,2-diphenyl-1-picylhydrazyl (DPPH) and 2,2-azino-bis(3-ethylbenzthiazoline)-6-sulfonic acid (ABTS) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). Acetonitrile, methanol, n-hexane, and isopropanol (HPLC grade) were purchased from Sigma-Aldrich Co., Ltd. (Saint Louis, MO, USA). Alkaline protease (enzyme activity >200,000 U/g, optimal pH 8.5~10.5, optimal temperature 40~55 °C), bicinchoninic acid (BCA) protein assay kit, bovine serum albumin (BSA) and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (trolox) standards were purchased from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). Food grade chitinase (enzyme activity 100,000 U/g, optimum PH 5~6, optimum temperature 45~60 °C) was purchased from Changmao Biochemical Engineering Co., Ltd. (Changzhou, China). Glucose standard and other reagents were purchased from Sinopharm Chemical Reagent Co., Ltd. (Beijing, China).

Methanol (Optima grade), formic acid (Optima grade), ammonium acetate, hexane, acetonitrile, and ammonium hydroxide were purchased from Fisher Scientific (Fair Lawn, NJ, USA). Oxalyl chloride, oleic acid, erucic acid, petroselaidic acid, heptane HPLC grade, methyl-tert-butyl-ether HPLC grade, isopropanol HPLC grade, acetic acid, and anhydrous dichloromethane were from Sigma Aldrich (St Louis, MO, USA). N, N-dimethylformamide, heptadecanoic acid, and eicosanoic acid were purchased from Aldrich Chemical Company (Milwaukee, WI, USA). Lauric acid, myristic acid, palmitic acid, stearic acid, and docosanoic acid were purchased from Acros Organics (New Jersey, USA). Elaidic acid was from MP Biomedical Inc. (Solon, OH, USA) and linoleamide was purchased from Enzo Life Sciences (Ann Arbor, MI, USA). Stearoyl ethanolamine, oleoylglycine, linoleoylglycine, palmitoylglycine, arachidonoylglycine, and arachidoylglycine were purchased from Cayman Chemicals (Ann Arbor, MI, USA). 1 monopalmitoyl-rac-glycerol (MAG) and tristearin (TAG) were from Sigma (St. Louis, MO) 99% purity and 1,2-dipalmitoyl-rac-glycerol (DAG) was from MP Biomedicals (Solon, Ohio).

All chemicals were of analytical reagent grade. The hydrochloric acid (37%), sodium oxalate (≥ 99.5%), deuterium oxide (99%D) and sodium azide (≥ 99.0%) were purchased from Sigma-Aldrich (Milan, Italy), while sodium salt of (trimethylsilyl)-propionic-2,2,3,3-d4 acid (TSP, 99%D) was purchased from Armar Chemicals (Döttingen, Switzerland). Acetonitrile and methanol LC/MS grade and isopropanol HPLC grade were purchased from Sigma-Aldrich (Milan, Italy). Water was doubly deionised (resistivity: 18 MΩ cm) with a Milli-Q water purification system (Merck Millipore, Darmstadt, Germany).

Mass Photometry experiments were performed on a Refeyn TwoMP (Refeyn Ltd) (49 (link)). No.1.5, 24 mm × 50 mm microscope coverslips (Thorlabs Inc) were cleaned by serial rinsing with Milli-Q water and HPLC-grade isopropanol (Sigma Aldrich), on which a CultureWell gasket (Grace Bio-labs) was then placed. All measurements were performed at 25 °C in Dulbecco's phosphate-buffered saline without calcium and magnesium (Thermo Fisher). For each measurement, 15 μl of Dulbecco's phosphate-buffered saline buffer was placed in the well for focusing, after which 3 to 5 μl of 100 nM protein was introduced and mixed. Movies were recorded for 60 s at 50 fps under standard settings. Mass Photometry measurements were calibrated using protein standard mixture: β-Amylase (56, 112 and 224 kDa) and Thyroglobulin (670 kDa). Mass Photometry data were processed using DiscoverMP (Refeyn Ltd).
Generation of Pde6a^C857S/C857S mice is described in Supporting information text and Fig. S5. All experimental procedures involving the use of mice were performed in accordance with the National Institutes of Health guidelines and the protocol approved by the University of Iowa Animal Care and Use Committee.

Mass photometry (MP) experiments were performed on a Refeyn TwoMP mass photometer (Refeyn Ltd). Microscope coverslips (24 × 50 mm, Thorlabs Inc.) were cleaned by serial rinsing with Milli-Q water and HPLC-grade isopropanol (Sigma-Aldrich) followed by drying with a filtered air stream. Silicon gaskets (Grace Bio-Labs) to hold the sample drops were cleaned in the same procedure immediately before measurement. All MP measurements were performed at room temperature using Dulbecco’s PBS (DPBS) without calcium and magnesium (ThermoFisher). The instrument was calibrated using a protein standard mixture: β-amylase (Sigma-Aldrich, 56, 112, and 224 kDa), and thyroglobulin (Sigma-Aldrich, 670 kDa). Before each measurement, 15 μl of DPBS buffer was placed in the well to find focus. The focus position was searched and locked using the default droplet-dilution autofocus function after which 5 μl of protein was added and pipetted up and down to briefly mix before movie acquisition was promptly started. Movies were acquired for 60 s (6000 frames) using AcquireMP (version 2.3.0; Refeyn Ltd) using standard settings. All movies were processed and analyzed using DiscoverMP (version 2.3.0; Refeyn Ltd).