Cellular BCAA content was measured with Branched Chain Amino Acid Assay Kit (abcam, Waltham, MA, USA) and followed the manufacturer’s protocol. Briefly, Cells were plated into six-well plates in triplicate per cell line. 24 h after seeding, cells were washed twice with pre-warmed HBSS and then pre-incubated with pre-warm HBSS for 2 min. Cells were then incubated with pre-warmed DMEM for 2 h then homogenized and centrifuged to remove cell debris and insoluble materials. Each sample with assay buffer was incubated for 30 min then measured optical density at 450 nm in a microplate reader. BCAA concentrations of test samples were calculated by standard curve (from 0 to 10 nmol) and corrected background by subtracting the value derived from the zero BCAA standards. These measurements were normalized to cellular protein content measured by BCA assay with the remaining lysate of each sample.
Branched chain amino acid assay kit
Manufactured by Abcam
Sourced in United States
The Branched Chain Amino Acid Assay Kit is a colorimetric assay used for the quantitative determination of branched-chain amino acids (leucine, isoleucine, and valine) in various samples. The kit provides a simple, straightforward method to measure the concentration of these amino acids.
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Lab products found in correlation
Enzyme immunoassay kit, by Arbor Assays (1 mentions)
Rat mouse insulin elisa, by Merck Group (1 mentions)
Colorimetric assay, by Cell Biolabs (1 mentions)
Picoprobe fructose fluorometric assay kit, by Abcam (1 mentions)
Hr series nefa hr 2 kit, by Fujifilm (1 mentions)
Insulin elisa kit, by ALPCO (1 mentions)
5 protocols using branched chain amino acid assay kit
1
Cellular BCAA Quantification Protocol
2
Biochemical Biomarker Measurement Protocol
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Serum norepinephrine was measured using a Norepinehprine Research ELISA kit (Rocky Mountain Diagnostics, Inc.), and corticosterone was measured using an enzyme immunoassay kit (Arbor Assays) according to the manufacturers’ protocols. Serum glucose was measured using a Glucose (oxidase) Reagent set, total cholesterol was measured using an Enzymatic Liquid kit, and triglycerides were measured using a GPO Trinder kit (all from TECO Diagnostics). High-density lipoprotein (HDL) and low-density lipoprotein (LDL) cholesterol were measured using automated reagents (Sekisui Diagnostics). Serum free fatty acids (FFAs) were measured using a colorimetric assay (Cell Biolabs, Inc.). The cholesterol and FFA assays were modified for use on a Konelab Arena 30 system (Thermo LabSystems Inc.). Plasma insulin was detected using a Rat/Mouse Insulin ELISA (Millipore), and branched chain amino acid plasma levels were detected using a Branched Chain Amino Acid Assay kit (Abcam).
3
Quantifying Branched-Chain Amino Acids
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BCAA concentration in serum and liver tissue was determined using the Branched Chain Amino Acid Assay Kit (ab83374, Abcam), in accordance with the manufacturer's protocol. For tissue samples, 500 μl of the cold assay buffer were added to 10 mg of the liver. Using a Dounce homogenizer on ice, the tissues were lysed. Samples were spun down, and the supernatant was collected.
4
Zebrafish Metabolite Profiling After Starvation
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Twelve hours after starvation, zebrafish blood was collected as described in our previous study25 ,30 . Contents of the gut were collected and homogenized in D-PBS at five-times the original volume. Then, the supernatants were obtained by centrifugation at 15,000 g for 10 min at 4 °C. Plasma and gut fructose levels were quantified using PicoProbe Fructose Fluorometric Assay Kit (Biovision, Mountain View, CA, USA), according to manufacturer’s instruction. Branched chain amino acids were quantified using Branched Chain Amino Acid Assay Kit (Abcam, Cambridge, MA, USA), according to manufacturer’s instruction.
5
Metabolic Profiling in Hypertensive Rats
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Blood glucose was measured in tail vein blood of SHR and WKY rats after a 6 hour fast using a glucometer (ACCU‐CHEK Nano, Bayer). Rats were then anesthetized with Inactin as described above and blood samples collected through a PE‐50 catheter inserted into the right carotid artery. Hearts were harvested and processed for metabolite analysis by Metabolon Inc. (see Data S1 ). Serum was stored at −80°C for subsequent measurements of free fatty acids (FFA), insulin, and branched chain amino acids (BCAA) using the HR Series NEFA‐HR(2) kit (Wako), insulin ELISA kit (80‐INSHU‐E01.1, ALPCO) and Branched Chain Amino Acid Assay kit (ab83374, ABCAM), respectively.
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