Organelle Isolation & Analysis Kits

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For the isolation of an enriched, functional mitochondrial fraction from animal tissue

This kit features specially formulated extraction reagents and optimized protocols for the preparation of mitochondrial fractions from either soft (liver or brain) or hard (skeletal or heart muscle) tissue. In addition, the integrity of the inner membrane of the resulting mitochondria is analyzed by measuring the uptake of the fluorescent carbocyanine dye (JC-1) which is supplied in the kit (Mitochondrial Isolation Kit Data). The integrity of the mitochondrial outer membrane may be assessed by measuring cytochrome c oxidase activity using the Cytochrome c Oxidase Assay Kit (CYTOC-OX1). The resulting intact mitochondrial preparation is suitable for apoptosis studies, 2D analysis for proteomics and many other applications. Each kit is sufficient for the extraction of mitochondria from 10 to 20 grams of animal tissue. Enough JC-1 staining solution is provided to perform 50 JC-1 assays of 2 mL each.

Features & Benefits

• Specially formulated extraction reagents & a proven procedure suitable for small scale isolation - Isolate an enriched, intact mitochodrial fraction in a microfuge tube.
• Produces functionally active, intact mitochondria - Resulting mitochondria are suitable for in vitro assays for apoptosis, oxidative stress or other studies.
• Includes stain & protocol for determining inner membrane integrity - Determine the integrity of the inner mitochondrial membrane without the need to purchase other reagents.
• Compatible with the Cytochrome c Oxidase Assay Kit - Allows easy determination of the integrity of the outer mitochondrial membrane

Components
  Extraction Buffer A, 5x
  Extraction Buffer B, 5x
  Storage Buffer, 5x
  Albumin Solution
  JC-1 Stain
  JC-1 Assay Buffer, 5x
  Trypsin

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For the determination of cytochrome c oxidase activity in soluble & membrane bound mitochondrial samples.

Cytochrome c oxidase is located on the inner mitochondrial membrane dividing the mitochondrial matrix from the intermembrane space. The Cytochrome c Oxidase Kit uses an optimized colorimetric assay based on observation of the decrease in absorbance of ferrocytochrome c measured at 550 nm, which is caused by its oxidation to ferricytochrome c by cytochrome c oxidase. This kit is suitable for the detection of mitochondrial outer membrane integrity and for the detection of mitochondria in subcellular fractions. (Cytochrome C Data). The Cytochrome c Oxidase Kit provides sufficient reagent for 100 tests.

Features & Benefits

• Simple, optimized protocol - Obtain reproducible results without the need for special training.
• Useful for determination of cytochrome c activity from any mitochondrial source - The enzyme is present in all mitochondria regardless of species.
• Useful for detecting the presence of mitochondria in subcellular fractions - Save time & increase confidence in the quality of organelle preparations.
• May be used in conjunction with the Mitochondrial Isolation Kit (MITO-ISO1) - Standardized mitochondrial preparation & analysis ensure reproducible results.

Components
  Assay Buffer 5x
  Enzyme Dilution Buffer 2x
  Cytochrome c
  Sodium Hydrosulfite
  Cytochrome c Oxidase (positive control)
  n-Dodecyl b-D-Maltoside

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For the isolation of intact endoplasmic reticulum from mammalian soft tissues & cultured cell lines by centrifugal separation

The endoplasmic reticulum (ER) is a network of interconnected tubules, vesicles, and sacs. They play a role in specialized cellular functions including protein synthesis, sequestering of calcium, production of steroids, storage and production of glycogen, and insertion of membrane proteins. The kit contains all the reagents required for ER preparations with various degrees of purity: crude ER (microsomes), Ca²⁺ precipitated rough ER (RER) enriched microsomes, and density gradient purified rough ER (RER) and smooth ER (SER). The ER isolation process can be monitored by measuring the activity of NADPH cytochrome c reductase Cytochrome c Reductase Assay Kit, (Product Code CY0100). The enzyme is an ER membrane protein that is commonly used as an ER marker. The isolated ER can be used for studying the cytochrome P450 system and xenobiotic metabolism, for studying lipid metabolism, and for the recovery of ER membrane and lumenal proteins. The kit was tested with rat liver, kidney, and brain; mouse liver; rabbit liver, kidney, spleen, heart, and brain; and with Jurkat and HeLa cell lines.

Features & Benefits

• Specially formulated extraction reagents & a proven procedure suitable for small scale isolation - Isolate an enriched, intact endoplasmic reticulum fraction in a microfuge tube.
• Produces functionally active, intact endoplasmic reticulum(ER) - Resulting ER are suitable for in vitro assays for profiling, lipd metabolism, P450 studies & membrane protein analysis
• Includes stain & protocol for determining inner membrane integrity - Determine the integrity of the endoplasmic reticulum membrane to confirm functional performance.
• Compatible with the Cytochrome c Reductase Assay Kit, (CY0100) - Allows easy determination of the integrity of the outer endoplasmic reticulum membrane.

Components
  Isotonic Extraction Buffer 5X
  Hypotonic Extraction Buffer
  Calcium Chloride Solution
  OptiPrep™ Density Gradient Medium
  Needle, 4 inch, 20 gauge

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For the isolation of intact chloroplasts from plant leaves

This kit enables isolation of chloroplasts through mechanical cell wall & cell membrane breakage, removal of cell debris & unbroken leaf tissue by filtration, collection of total cell chloroplast fraction by centrifugation, & separation of intact chloroplasts (Data) from broken chloroplasts using a Percoll® layer or gradient. (Chloroplast Isolation Kit Data) Sigma's Chloroplast Isolation Kit has been tested for use with spinach, pea, lettuce, cabbage, mangold and tobacco.

Features & Benefits

• Specifically formulated buffer - Saves time using easy-to-follow, simplified protocol based on proven methods.
• Isolates intact chloroplasts - Useful for studies of such processes as carbon assimilation, electron flow and phosphorylation, metabolic transport, or protein targeting.
• Kits are useful for both proteomic or genomic applications - Provides optimized conditions for isolation of intact chloroplasts from leaves of plants commonly used for research.

Components
  Chloroplast Isolation Buffer 5x (CIB)
  Percoll®
  Bovine Serum Albumin (BSA)
  Filter Mesh 100

Kits for Nuclei Enrichment

• NUC101
• NUC201

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For the determination of cytochrome c oxidase activity in tissue and cell samples

NADPH-cytochrome c reductase is widely used as an endoplasmic reticulum marker and biomarker for ecological pollution and dietary lipid uptake. This kit is a fast and convenient analysis of NADPH-cytochrome c reductase activity in cell or tissue extracts and in purified microsomes [endoplastic reticulum (ER)]. It is suitable for samples prepared from mammalian tissues such as liver, kidney, brain, spleen, and heart muscle from several species, as well as on lysates from cell lines such as HeLa, HepG2, and Jurkat. Additionally, it has been tested on yeast strains P. pastoris and S. cerevisiae samples.

Features & Benefits

• Detection of endoplasmic reticulum (ER) during isolation and subcellular fractionation by density gradient separation - Validates degree of enrichment and can be multiplexed to determine purity of enriched fraction against other organelles.
• Assaying NADPH-cytochrome c reductase levels in various sources after intoxication by drugs or challenge by xenobiotics - Determine and compare the results of environmental perturbations.

Components
  Assay Buffer: 100 mL
  Enzyme Dilution Buffer: 25 mL
  Cytochrome c Reductase [NADPH] Positive Control: 50 µL
  Cytochrome c Oxidase Inhibitor Solution: 1 mL
  Cytochrome c: 50 mg
  NADPH: 25 mg

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For the enrichment of intact peroxisomes from tissues and cells

Isolated peroxisomes are used for studying lipid b-oxidation, amino acid metabolism and biosynthesis of ether-linked glycerolipids and bile acids.This kit provides all the necessary reagents and a detailed protocol for the isolation of highly purified peroxisomes from animal tissues and cells, by differential density gradient centrifugation using iodixanol [Optiprep] density gradient centrifugation (enrichment data). The kit has been used for preparation of peroxisomes from rat liver, rat kidney and rabbit liver as well as HEK293 and HepG2 cells.

Features & Benefits

• Specially formulated extraction reagents for research scale applications - Save time and minimize waste.
• Produces functional intact organelles - Resulting peroxisomes are suitable for metabolic assays and disease state analysis.
• Compatible with products for structure confirmation - Easily confirm intactness with companion kit test for cytochrome C reductase.

Components
  Peroxisome Extraction Buffer 5X
  Optiprep Dilution Buffer
  Optiprep Density Gradient Medium
  Protease Inhibitor Cocktail for mammalian cells and tissues

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For the determination of catalase activity in tissues and cells

A simple and easy colorimetric assay for analysis of peroxisome enrichment and catalase activity in various tissues. Catalase is a ubiquitous antioxidant enzyme in mammalian and non-mammalian aerobic cells containing a cytochrome system. Catalase provides protection against oxidative damage to living cells and it's role in oxidative stress related diseases has been widely studied.

Components
  Assay Buffer 10x: 100 mL
  Chromogen Reagent: 1 vial
  Stop Solution: 100 mL
  Catalase Positive Control: 0.25 mL
  3% Hydrogen peroxide: 10 mL
  Peroxidase horseradish (Sigma P6782): 5 mg
  Enzyme Dilution Buffer: 100 mL

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