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Aconitase assay kit

Manufactured by Cayman Chemical
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The Aconitase Assay Kit is a laboratory tool designed to measure the activity of the enzyme aconitase, which is involved in the citric acid cycle. The kit provides the necessary reagents and protocols to quantify the enzymatic activity of aconitase in various sample types.

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Aconitase

21 protocols using aconitase assay kit

1

Aconitase Activity Quantification

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Aconitase activities were determined using the Aconitase Assay Kit (Cayman Chemical Company, 705502). Cell protein lysates (50 μl) were added to 200 μl of substrate mix [50 mM Tris/HCl pH 7.4, 0.4 mM NADP, 5 mM Na citrate, 0.6 mM MgCl2, 0.1% (v/v) Triton X-100 and 1 U isocitrate dehydrogenase] and the reactions were incubated at 37°C for 15 minutes, followed by spectrophotometric absorbance measurements at 340 nm at 37°C every minute for 15 minutes and subsequent determination of the reaction slope. Aconitase activities of mouse cells were then normalized to citrate synthase activities, which were determined using a citrate synthase assay kit (Sigma, CS0720).
Anjomani Virmouni S., Ezzatizadeh V., Sandi C., Sandi M., Al-Mahdawi S., Chutake Y, & Pook M.A. (2015). A novel GAA-repeat-expansion-based mouse model of Friedreich’s ataxia. Disease Models & Mechanisms, 8(3), 225-235.
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2

Hypoxia-Induced Oxidative Stress Assay

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Cells plated onto poly-lysine coated cover slips were exposed to 4days of CH. CM-H2DCFDA 5-(and-6) chloromethyl 2’, 7’dichlorodihydrofluorescein diacetate; 25uM) was added to the cells 30 min prior to terminating the hypoxic exposure. Subsequently, cells were washed, mounted on glass slides and fluorescence images were taken. Fluorescence intensity >5 times background was defined as positive staining and the number of cells with positive staining was pooled across five fields in a given specimen. Aconitase activity was measured using an aconitase assay kit (Cayman Chemical Company; # 705502) as described. Protein concentration was estimated in the cell extracts using Bio-Rad protein assay kit. The enzyme activities were expressed as nmol/mg of protein.
Vaddi D.R., Piao L., Khan S.A., Wang N., Prabhakar N.R, & Nanduri J. (2019). Hypoxia induced hERG trafficking defect linked to cell cycle arrest in SH-SY5Y cells. PLoS ONE, 14(4), e0215905.
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3

Aconitase Activity Measurement in Cells

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Whole-cell extracts were obtained by homogenization of cultured cells in 50 mM Tris–HCl, pH 8, 10% (v/v) glycerol, 5 mM EDTA, 150 mM KCl, 1 mM phenylmethylsulfonyl fluoride. Insoluble material was removed by centrifugation at 10 000 g at 4 °C for 10 min.42 (link) Aconitase activity was measured using an Aconitase Assay Kit (Cayman Chemical, Ann Arbor, MI, USA). 50 μl whole-cell lysates were added to 200 μl of substrate mix, (50 mM Tris/HCl pH 7.4, 0.4 mM NADP, 5 mM Na citrate, 0.6 mM MgCl2, 0.1% (v/v) Triton X-100 and 1 U isocitrate dehydrogenase) then the reaction was initiated by adding 50 μl of diluted substrate solution followed by incubation at 37 °C for 15 min. Absorbance was monitored by spectrophotometry every minute for 15 min at 340 nm 37 °C to determine the reaction slope. Aconitase activities of cells were then normalized to citrate synthase activities, which were determined using a citrate synthase assay kit (SIGMA, CS0720), as previously described.53 (link)
Khonsari H., Schneider M., Al-Mahdawi S., Chianea Y.G., Themis M., Parris C., Pook M.A, & Themis M. (2016). Lentivirus-meditated frataxin gene delivery reverses genome instability in Friedreich ataxia patient and mouse model fibroblasts. Gene Therapy, 23(12), 846-856.
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4

Aconitase Activity Assay in K562 Cells

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Aconitase activity from whole K562 cell extracts, obtained from 5×106 cells, was assayed with the Aconitase Assay Kit (Cayman Chemicals), per manufacturer’s instructions.
Ast T., Meisel J.D., Patra S., Wang H., Grange R.M., Kim S.H., Calvo S.E., Orefice L.L., Nagashima F., Ichinose F., Zapol W.M., Ruvkun G., Barondeau D.P, & Mootha V.K. (2019). Hypoxia rescues frataxin loss by restoring iron sulfur cluster biogenesis. Cell, 177(6), 1507-1521.e16.
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5

Aconitase Activity Quantification in Staphylococcus Cultures

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Whole cytosolic protein was isolated from 18 ml of aerobic TSB-G cultures grown for 3 hours. Addition of 15 µM thioridizine HCl was completed at time of inoculation for treated cultures. Cells were isolated by centrifugation at 3901 ×g for 10 minutes at 4 °C followed by washing with 1 ml of cold 1× PBS. Cell lysis was completed in 1× aconitase assay buffer (50 mM Tris-HCl, pH 7.4) containing 100 µg/ml lysostaphin (Sigma) and 27.3 Kunitz units DNase I (Qiagen). After 30 minutes of incubation at 37°C, cell debris was removed by centrifugation at 13,000 × g (4°C) and proteins were immediately frozen at −80°C for future analysis. Aconitase activity was quantified using the Cayman Chemicals Aconitase Assay Kit, following the manufacturer’s recommended protocols. Assays were performed on 1:4 dilutions of each protein sample, and optical density measurements at 340 nm were taken at 1 minute intervals for an hour with incubation at 37°C. No substrate controls were included to measure any background noise. Enzyme activity was determined by measuring the reaction rate (ΔA340/min.) and using the NADPH extinction coefficient (0.00313 µM−1, adjusted for the 0.503 cm path length of the well). All sample activity was normalized to total cytosolic protein as determined by the Pierce™ BCA protein quantification assay (Life Technologies).
Mogen A.B., Carroll R.K., James K.L., Lima G., Silva D., Culver J.A., Petucci C., Shaw L.N, & Rice K.C. (2017). Staphylococcus aureus nitric oxide synthase (saNOS) modulates aerobic respiratory metabolism and cell physiology. Molecular microbiology, 105(1), 139-157.
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6

Aconitase Activity Assay Protocol

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Aconitase activities were determined using the Aconitase Assay Kit (Cayman Chemical Company, 705502). To perform the assays, cultured cells were washed with cold PBS, scraped off the flask and pelleted by centrifugation at 800 g for 10 min. Cell pellets were suspended in cell lytic buffer (Cayman) for 15 min at 4 °C, then centrifuged at 10,000 g for 10 min, followed by BCA protein concentration determination and dilution of cell protein lysates to 0.5ug/µl in 1x assay buffer (Cayman). 50 µl of 0.5 ug/µl cell protein lysates were added to 200 µl of substrate mix (50 mM Tris/HCl pH 7.4, 0.4 mM NADP, 5 mM Na citrate, 0.6 mM MgCl2, 0.1% (v/v) Triton X-100 and 1 U isocitrate dehydrogenase) and the reactions were incubated at 37 °C for 15 min, followed by spectrophotometric 340 nm absorbance measurements every minute for 15 min at 37 °C to determine the reaction slope. Aconitase activities of samples were then normalized to citrate synthase activities, which were determined using a citrate synthase assay kit (Sigma, CS0720).
Mikaeili H., Sandi M., Bayot A., Al-Mahdawi S, & Pook M.A. (2018). FAST-1 antisense RNA epigenetically alters FXN expression. Scientific Reports, 8, 17217.
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7

Measuring Glutathione and Aconitase

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Total glutathione (GSH + GSSG) and GSSG levels were measured as previously described. Aconitase activity was measured with an Aconitase Assay Kit (Cayman Chemical, Ann Arbor, MI, USA) according to the manufacturer's protocol. The methods are described as additional experimental procedures in Data S1 (Supporting information).
Fujii N., Narita T., Okita N., Kobayashi M., Furuta Y., Chujo Y., Sakai M., Yamada A., Takeda K., Konishi T., Sudo Y., Shimokawa I, & Higami Y. (2017). Sterol regulatory element‐binding protein‐1c orchestrates metabolic remodeling of white adipose tissue by caloric restriction. Aging Cell, 16(3), 508-517.
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8

Aconitase Activity Assay in FRDA Tissues

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Aconitase activities of YG8JR and Y47JR heart and cerebellum tissues (n = 8 each group) were determined using the Aconitase assay kit (Cayman Chemical Company, MI, United States) and normalized to citrate synthase activities determined using a Citrate Synthase assay kit (Sigma, MO, United States), as previously described (Sandi et al., 2014 (link); Anjomani Virmouni et al., 2015b (link)). All experiments were performed in triplicates and protein concentrations were determined using Pierce BCA assay (Thermo Fisher). The absorbance detection was carried out using CLARIOstar Microplate Reader (BMG LABTECH, Aylesbury, United Kingdom).
Kalef-Ezra E., Edzeamey F.J., Valle A., Khonsari H., Kleine P., Oggianu C., Al-Mahdawi S., Pook M.A, & Anjomani Virmouni S. (2023). A new FRDA mouse model [Fxnnull:YG8s(GAA) > 800] with more than 800 GAA repeats. Frontiers in Neuroscience, 17, 930422.
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9

Aconitase Activity in Fh1-Deficient Cells

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The activity of aconitase in Fh1-deficient cells was measured using the Aconitase Assay Kit (Cayman), following the manufacturer’s instruction.
Tyrakis P.A., Yurkovich M.E., Sciacovelli M., Papachristou E.K., Bridges H.R., Gaude E., Schreiner A., D’Santos C., Hirst J., Hernandez-Fernaud J., Springett R., Griffiths J.R, & Frezza C. (2017). Fumarate Hydratase Loss Causes Combined Respiratory Chain Defects. Cell Reports, 21(4), 1036-1047.
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10

Aconitase Activity Quantification

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Aconitase activities were determined using the Aconitase Assay Kit (Cayman Chemical Company, 705502). To perform the assays, cell protein lysates (50 µl) were added to 200 µl of substrate mix (50 mM Tris/HCl pH 7.4, 0.4 mM NADP, 5 mM Na citrate, 0.6 mM MgCl2, 0.1% (v/v) Triton X-100 and 1 U isocitrate dehydrogenase) and the reactions were incubated at 37°C for 15 min, followed by spectrophotometric absorbance measurements every minute for 15 min at 340 nm 37°C to determine the reaction slope. Aconitase activities of mouse cells were then normalized to citrate synthase activities, which were determined using a citrate synthase assay kit (Sigma, CS0720).
Sandi C., Sandi M., Jassal H., Ezzatizadeh V., Anjomani-Virmouni S., Al-Mahdawi S, & Pook M.A. (2014). Generation and Characterisation of Friedreich Ataxia YG8R Mouse Fibroblast and Neural Stem Cell Models. PLoS ONE, 9(2), e89488.
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