Left ventricular tissue was homogenized in PBS on ice and centrifuged at 4°C, at 10,000 rpm for 10 min. The supernatant was collected for measurements. As descripted in the manufacturer’s protocol and in our studies (El-Mas et al., 2012 (link), Ibrahim et al., 2014 (link), Yao and Abdel-Rahman, 2016 (link)), 5 μg of sample protein was used to measure the catalytic activity by colorimetric catalase assay kit (Sigma-Aldrich, St. Louis, MO). The absorbance was read at 520 nm. A mitochondrial ALDH2 activity assay kit (Abcam, Ann Arbor, MI) was used for the measurement of mitALDH2 activity in 500 μg sample protein; the absorbance was read at 450 nm every 3 min for 1 hr.
Mitochondrial aldh2 activity assay kit
Manufactured by Abcam
Sourced in United Kingdom
The Mitochondrial ALDH2 activity assay kit is a laboratory tool designed to measure the activity of the enzyme Aldehyde Dehydrogenase 2 (ALDH2) in mitochondria. The kit provides a quantitative colorimetric assay to determine ALDH2 activity levels.
Automatically generated - may contain errors
Lab products found in correlation
Colorimetric catalase assay kit, by Merck Group (2 mentions)
Colorimetric cat assay kit, by Merck Group (1 mentions)
Cleaved caspase 3 antibody, by Cell Signaling Technology (1 mentions)
Revertaid rt reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Nec 1, by Merck Group (1 mentions)
Gapdh antibody, by Boster Bio (1 mentions)
Dhe fluorescent probe, by Merck Group (1 mentions)
Alda 1, by Merck Group (1 mentions)
Aldh2 antibody, by Santa Cruz Biotechnology (1 mentions)
Sybr premix dimer eraser perfect real time, by Takara Bio (1 mentions)
Gapdh, by Sangon (1 mentions)
6 protocols using mitochondrial aldh2 activity assay kit
1
Enzymatic Activity Assay for Left Ventricular Tissue
2
Quantification of ALDH2 and Catalase Activity
3
Evaluation of Catalase and ALDH2 Activity
4
Alda-1 and Necroptosis Inhibition Protocol
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Alda-1, the specific activator of ALDH2, necrostatin-1 (Nec-1), the specific inhibitor of necroptosis and the dihydroethidium (DHE) fluorescent probe were obtained from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). The mitochondrial ALDH2 activity assay kit was obtained from Abcam (Cambridge, UK). The ALDH2 antibody was obtained from Santa Cruz Biotechnology, Inc., (Dallas, TX, USA). The RIP1 and RIP3 antibodies were obtained from Abcam. The mixed lineage kinase domain like pseudokinase (MLKL) antibody was obtained from Affinity Biosciences (Cambridge, UK). The cleaved caspase-3 antibody was obtained from Cell Signaling Technology, Inc., (Danvers, MA, USA). The GAPDH antibody was acquired from Boster Biological Technology Co., Ltd. (Wuhan, China). A Cell Counting Kit-8 (CCK-8) assay kit was from Bestbio Life Technology (Shanghai, China; http://www.bestbio.com.cn ; cat. no. BB-4202-1). Primers for ALDH2, RIP1, RIP3, MLKL and GAPDH were acquired from Sangon Biotech Co., Ltd. (Shanghai, China; sequences are presented in Table I ). A RevertAid RT Reverse Transcription kit was purchased from Thermo Fisher Scientific, Inc., (Waltham, MA, USA). The SYBR® Premix DimerEraser™ (Perfect Real Time) was acquired from Takara Bio, Inc. (Otsu, Japan).
5
ALDH2 Activity Assay in Rat Aorta
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ALDH2 activity was determined in mitochondria isolated from rat thoracic aorta following solubilization and extraction as specified in the manufacturer's recommendations (mitochondrial ALDH2 activity assay kit; Abcam, Cambridge, UK). The homogenate was then incubated on ice for 20 min and centrifuged at 16 000× g for 20 min at 4°C. Protein concentration of the supernatant was determined and 20 μg of protein was used to detect ALDH2 activity. In this assay, the generation of NADH is coupled to the 1:1 reduction of a reporter dye to yield reaction product concentration, which was monitored by measuring the absorbance increase at 450 nm.
6
Mitochondrial ALDH2 Activity Assay
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The mitochondrial ALDH2 activity in cell lysates was detected by mitochondrial ALDH2 activity assay kit (Abcam, Cambridge, UK) according to the manufacturer's instructions. This assay kit is based on the ability of ALDH2 to catalyse the substrate NAD+ to produce NADH, which has strong absorption at 450 nm.
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