Micro bca protein assay kit

All experiments on biological materials were performed in accordance with the ethical standards of the institutional research committee and with the 1964 Helsinki Declaration and its later amendments. In keeping with ethical guidelines, all blood and cell samples were obtained for storage and analyzed only after written informed consent of the patients (and/or their family members) was obtained, using a form approved by the local Ethics Committee (Project ID code: 16774_bio, 5 May 2020, Comitato Etico Regionale per la Sperimentazione Clinica della Regione Toscana, Area Vasta Centro, Florence, Italy). Controls and patients’ samples were anonymized and used only for research purposes.
The new compounds were screened at 1 mM concentration towards β-galactosidase (β-Gal) and β-glucosidase (GCase) in leukocytes isolated from healthy donors (controls). Isolated leukocytes were disrupted by sonication, and a Micro BCA Protein Assay Kit (Sigma–Aldrich, St. Louis, MO, USA) was used to determine the total protein amount for the enzymatic assay, according to the manufacturer’s instructions.

All experiments on biological materials were performed in accordance with the ethical standards of the institutional research committee and with the 1964 Helsinki Declaration and its later amendments. In keeping with ethical guidelines, all blood and cell samples were obtained for storage and analysed only after written informed consent of the patients (and/or their family members) was obtained, using a form approved by the local Ethics Committee (Codice Protocollo: Lysolate “Late onset Lysosomal Storage Disorders (LSDs) in the differential diagnosis of neurodegenerative diseases: development of new diagnostic procedures and focus on potential pharmacological chaperones (PCs). Project ID code: 16774_bio, 5 May 2020, Comitato Etico Regionale per la Sperimentazione Clinica della Regione Toscana, Area Vasta Centro, Florence, Italy).
Controls and patients’ samples were anonymized and used only for research purposes.
The new compounds were screened at 1 mM concentration towards β-Galactosidase (β-Gal) and β-Glucosidase (GCase) in leukocytes isolated from healthy donors (controls).
Isolated leukocytes were disrupted by sonication, and a Micro BCA Protein Assay Kit (Sigma–Aldrich, St. Louis, MO, USA) was used to determine the total protein amount for the enzymatic assay, according to the manufacturer’s instructions.

The compound CV82 was screened at a 1 mM concentration towards β-Galactosidase (β-Gal) in leukocytes isolated from healthy donors (controls). Isolated leukocytes were disrupted by sonication, and a Micro BCA Protein Assay Kit (Sigma-Aldrich, St. Louis, MO, USA) was used to determine the total protein amount for the enzymatic assay, according to the manufacturer’s instructions. β-Gal activity was measured in a flat-bottomed 96-well plate. Compound-CV82 solution (3 μL), 4.29 μg/μL of leukocyte homogenate 1:10 (7 μL), and substrate 4-methylumbelliferyl β-d-Galactopyranoside (1.47 mM, 20 μL, Sigma-Aldrich) in acetate buffer (0.1 M, pH 4.3) containing NaCl (0.1 M) and sodium azide (0.02%) were incubated at 37 °C for 1 h. The reaction was stopped by the addition of sodium carbonate (200 μL; 0.5M, pH 10.7) containing Triton X-100 (0.0025%), and the fluorescence of 4-methylumbelliferone released by β-Gal activity was measured in a SpectraMax M2 microplate reader (λex = 365 nm, λem = 435 nm; Molecular Devices, San Jose, CA, USA). The percentage of β-Gal inhibition is given with respect to the control (Ctrl, without compound). Data are mean ± S.D. (n = 3).

Cell lysate was prepared in RIPA lysis buffer. The protein concentration was determined with Micro BCA™ protein assay kit (Millipore, MA, USA). 35 μg protein was loaded into acrylamide gels and then transferred onto polyvinylidene fluoride membranes (Amersham Biosciences, Piscataway, NJ) after electrophoresis. The membranes were incubated with horseradish peroxidase-conjugated secondary antibody and probed with ECL western blotting detection system (Millipore, MA, USA) and visualized with the BioSpectrum AC imaging system.

Cell lysate was harvested in RIPA lysis buffer and the amount of protein was determined with Micro BCA Protein Assay kit (Millipore, MA, USA). 30μg protein lysate was loaded into acrylamide gels and then transferred onto polyvinylidene fluoride membranes (Amersham Biosciences, Piscataway, NJ). The membrane was blocked with 5% nonfat dry milk and incubated with primary antibody for specific protein overnight. The membranes were incubated with horseradish peroxidase-conjugated secondary antibody and probed with ECL western blotting detection system (Millipore, MA, USA) and visualized with the BioSpectrum AC imaging system. The catalog number of antibody was as the following: the anti-ACSL1 antibody (#4047, Cell Signaling) and the anti-β-actin antibody (GTX109639, GeneTex).