Apoalert cell fractionation kit

Manufactured by Takara Bio

Sourced in United States

The ApoAlert Cell Fractionation Kit is a laboratory equipment designed for the isolation and separation of cellular organelles and subcellular components. It provides a reliable and efficient method for the extraction and purification of specific organelles, such as mitochondria, nuclei, and cytosol, from mammalian cells.

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Lab products found in correlation

Doxorubicin, by Bio-Techne (1 mentions) Anti mtor antibody, by Cell Signaling Technology (1 mentions) Anti β actin, by Santa Cruz Biotechnology (1 mentions) Anti survivin, by Cell Signaling Technology (1 mentions) Anti gapdh, by Santa Cruz Biotechnology (1 mentions) Pf4708671, by Santa Cruz Biotechnology (1 mentions) Anti phospho mtor ser2481, by Cell Signaling Technology (1 mentions) Anti bcl 2, by Abcam (1 mentions) Insulin, by Merck Group (1 mentions) Anti bax, by Abcam (1 mentions) Actin, by Merck Group (1 mentions)

8 protocols using apoalert cell fractionation kit

Measuring Mitochondrial Dysfunction

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The loss of mitochondrial potential was determined using mitochondrial potential sensor JC-1 (Molecular Probes, Invitrogen). Control or cells treated with PM (1×10⁶) were loaded with mitochondrial sensor JC-1 dye (10 μg/ml) for 10 min at 22°C. Cells were then analyzed by flow cytometry. In normal cells, dye is aggregated in mitochondria, fluoresces red, and is detected in the FL2 channel. In cells with altered mitochondrial potential, the dye fails to accumulate in the mitochondria, remains as monomers in the cytoplasm, fluoresces green and is detected in the FL1 channel.
For effect on mitochondrial cytochrome c, mitochondrial and cytosolic fractions of control and treated cells were prepared using ApoAlert Cell Fractionation Kit (Clontech Laboratories) and were separated on a 14% SDS-PAGE gel. After transfer of proteins, membranes were probed with cytochrome c antibody.

DEEB D., GAO X., LIU Y.B., PINDOLIA K, & GAUTAM S.C. (2014). Pristimerin, a quinonemethide triterpenoid, induces apoptosis in pancreatic cancer cells through the inhibition of pro-survival Akt/NF-κB/mTOR signaling proteins and anti-apoptotic Bcl-2. International Journal of Oncology, 44(5), 1707-1715.

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Measuring Cytochrome c Release Fractions

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For measuring the cytochrome c release, the cytosol and mitochondrial fractions were prepared as described previously [17 (link)]. Briefly, cells were harvested by centrifugation at 600 ×g for 10 min at 4 °C. The cell pellets were washed once with ice-cold PBS and resuspended with five volumes of buffer A (20 mM Hepes-KOH, pH 7.4, 10 mM KCl, 1.5 mM MgCl₂, 1 mM sodium EDTA, 1 mM sodium EGTA, 1 mM dithiothreitol, and 0.1 mM phenylmethylsulfonyl fluoride) containing 250 mM sucrose. The cells were homogenized with 10 strokes of a Teflon homogenizer, and the homogenates were centrifuged twice at 750 ×g for 10 min at 4 °C. The supernatants were centrifuged at 10,000 ×g for 15 min at 4 °C, and the resulting mitochondria pellets were resuspended in buffer A containing 250 mM sucrose and frozen at –80 °C. The supernatants were further centrifuged at 16,000 ×g for 1 h at 4 °C, and the resulting supernatants were frozen at –80 °C for further experiments. After treatment with the indicated drugs, mitochondrial and cytosolic fractions were extracted from the cells using the ApoAlert Cell Fractionation Kit (Clontech, Mountain View, CA) according to the manufacturer’s instructions. Cytochrome c expression was determined using a monoclonal antibody through western blotting.

Zhu X., Xie M., Wang K., Zhang K., Gao Y., Zhu L, & Zhou F. (2014). The effect of puerarin against IL-1β-mediated leukostasis and apoptosis in retinal capillary endothelial cells (TR-iBRB2). Molecular Vision, 20, 1815-1823.

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Insulin and Doxorubicin Cytotoxicity Pathway

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Insulin, human recombinant from Saccharomyses cerevisiae, was purchased from Sigma-Aldrich. Doxorubicin was obtained from Tocris. Anti-survivin, anti-caspase-3 (cleaved form), anti-phospho-p53 (Ser¹⁵), anti-phospho-Akt (Ser⁴⁷³), anti-Akt, anti-phospho-mTOR (Ser²⁴⁸¹) and anti-mTOR antibodies were obtained from Cell Signaling. Anti-Sp1, anti-phospho-p70S6K (Thr⁴²¹/Ser⁴²⁴), anti-p70S6K, anti-β-actin, anti-GAPDH, anti-Smac/DIABLO antibodies and p70S6K inhibitor PF4708671 were purchased from Santa Cruz Biotechnology. Anti-VDAC1, anti-p53, anti-Bcl-2, and anti-BAX antibodies were obtained from Abcam. PI3K inhibitor LY294002 and mTOR inhibitor Rapamycin were purchased from Calbiochem. Anti-cytochrome c antibody was included in ApoAlert Cell Fractionation Kit (Clontech).

Lee B.S., Oh J., Kang S.K., Park S., Lee S.H., Choi D., Chung J.H., Chung Y.W, & Kang S.M. (2015). Insulin Protects Cardiac Myocytes from Doxorubicin Toxicity by Sp1-Mediated Transactivation of Survivin. PLoS ONE, 10(8), e0135438.

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Mitochondrial Fractionation from Tissue

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Tissue fractionation was performed using the ApoAlert™ Cell Fractionation kit (Clontech Laboratories Inc. Mountain View, CA, USA) according to the manufacturer’s instructions. Tissue fragments were cut into small pieces, washed with Wash buffer, suspended in Fractionation Buffer Mix containing DTT 1 mM and then homogenized with 65 strokes in a glass-Teflon homogenizer. Homogenate was centrifuged at 700× g for 10 min. The pellet was discarded and supernatant centrifuged at 10,000× g for 25 min. The pellet (Mitochondrial fraction) was resuspended in YY buffer (50 mM HEPES at pH 7.5, 10% glycerol, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 1 mM EGTA) containing 4.0% (w/v) of SDS. Supernatant (cytosolic fraction) was collected in a separate tube. The protein concentration of each fraction was determined using Bradford protein assay. Lysate of each fraction was then processed for Western blot. The mitochondrial TRAP-1 and cytosolic PP2A were used as fraction purity markers.

Solar Fernandez V., Fiocchetti M., Cipolletti M., Segatto M., Cercola P., Massari A., Ghinassi S., Cavaliere F, & Marino M. (2021). Neuroglobin: A New Possible Marker of Estrogen-Responsive Breast Cancer. Cells, 10(8), 1986.

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Mitochondrial Isolation from AGS Cells

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AGS cells were collected by a desired protocol after incubation with AS or JQ1 alone or in combination. The extraction and isolation of mitochondrial and cytoplasmic fractions were performed according to the ApoAlert^® Cell Fractionation Kit (Clontech, Shiga, Japan). The AGS cells were first centrifuged at 600× g for 5 minutes at 4°C to remove supernatant and then resuspended in 1 mL of ice-cold wash buffer. Next, cells were centrifuged at 600× g for another 5 minutes at 4°C and were then resuspended in 0.8 mL of ice-cold Fractionation Buffer Mix (2 µL Protease Inhibitor Cocktail+1 µL DTT+1 mL 1× Fraction Buffer) after displacing supernatant. After the incubation on ice for 10 minutes, cells were then homogenized 50 passes in an ice-cold tissue grinder. The homogenate was then transferred to a 1.5 mL microcentrifuge tube, followed by centrifugation at 700× g for 10 minutes at 4°C. After centrifugation, the supernatant was transferred to a new, 1.5 mL tube and was centrifuged at 10,000× g for 25 minutes at 4°C. Finally, the pellet was resuspended in 0.1 mL Fractionation Buffer Mix as the mitochondrial fraction, and the supernatant was collected as the cytosolic fraction.

Tan Z., Zhang X., Kang T., Zhang L, & Chen S. (2018). Arsenic sulfide amplifies JQ1 toxicity via mitochondrial pathway in gastric and colon cancer cells. Drug Design, Development and Therapy, 12, 3913-3927.

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Subcellular Fractionation for Protein Analysis

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Cell fractionation was performed using ApoAlert™ Cell Fractionation kit (Clontech Laboratories Inc. Mountain View, CA, USA) according to manufacturer’s instructions. After stimulation, cells were harvested with trypsin (1%, v/v), suspended with complete medium, and centrifuged at 600g for 5 min. Pellet was suspended in Fractionation Buffer Mix containing DTT 1 mM and homogenized in a Dounce tissue grinder. Homogenate was centrifuged at 700g for 10 min. Pellet was suspended in Fractionation Buffer Mix to obtain mitochondrial fraction. The mitochondrial TNF-receptor associated protein 1 (TRAP-1) and cytosolic Protein Phosphatase 2A (PP2A) markers were used as mitochondrial fraction purity indicators. Protein concentration of each fraction was determined using Bradford protein assay. Lysate of each fraction was then processed for Western Blot or used for immunoprecipitation assay.

Fiocchetti M., Cipolletti M., Leone S., Naldini A., Carraro F., Giordano D., Verde C., Ascenzi P, & Marino M. (2016). Neuroglobin in Breast Cancer Cells: Effect of Hypoxia and Oxidative Stress on Protein Level, Localization, and Anti-Apoptotic Function. PLoS ONE, 11(5), e0154959.

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Cytochrome c Release Quantification

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The cytosol and mitochondrial fractions were respectively collected as described previously to measure the release of cytochrome c [37 (link)]. After treatment, mitochondrial and cytosolic fractions were extracted from the cells using Apo Alert Cell Fractionation Kit (Clontech, Mountain View, CA, USA) according to the manufacturer’s instructions. Cytochrome c expression was analyzed using a monoclonal antibody through western blot analysis.

Wang K., Zhu X., Zhang K., Wu Z., Sun S., Zhou F, & Zhu L. (2016). Neuroprotective Effect of Puerarin on Glutamate-Induced Cytotoxicity in Differentiated Y-79 Cells via Inhibition of ROS Generation and Ca2+ Influx. International Journal of Molecular Sciences, 17(7), 1109.

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Cytosolic and Mitochondrial Fractionation

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Cells treated with etoposide or gemcitabine for 4 h were washed twice, and cytosolic and mitochondrial fractions were isolated, using Mitochondria Isolation Kit (Pierce, Rockford, IL, USA). Cytosolic fractions were probed for cytochrome c and Cox IV (a mitochondrial marker) with antibodies from the ApoAlert Cell Fractionation kit (Clontech, Mountain View, CA, USA), using actin (Sigma-Aldrich) as a loading control.

Radke J.R., Siddiqui Z.K., Figueroa I, & Cook J.L. (2016). E1A enhances cellular sensitivity to DNA-damage-induced apoptosis through PIDD-dependent caspase-2 activation. Cell Death Discovery, 2, 16076-.

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