Cytochrome C ELISA Kit

Catalog No. ABIN955774

Human Cytochrome C ELISA Kit, Colorimetric assay for quantification of Human Cytochrome C.

Quick Overview for Cytochrome C ELISA Kit (ABIN955774)

Target: Cytochrome C (CYCS) (Cytochrome C, Somatic (CYCS))

Reactivity: Human

Detection Method: Colorimetric

Method Type: Sandwich ELISA

Application: ELISA

Product Details Cytochrome C ELISA Kit

Purpose: The Human Cytochrome c ELISA is an enzyme-linked immunosorbent assay for the quantitative determination of human Cytochrome c in cell culture lysates.

Analytical Method: Quantitative

Specificity: The cross reactivity of circulating factors of the immune system was evaluated by spiking these proteins at physiologically relevant concentrations into a human Cytochrome c positive sample. There was no cross reactivity detected.

Sensitivity: The limit of detection of human Cytochrome c defined as the analyte concentration resulting in an absorption significantly higher than that of the dilution medium (mean plus 2 standard deviations) was determined to be 0.05 ng/mL (mean of 6 independent assays).

Characteristics: An anti-human Cytochrome c coating antibody is adsorbed onto microwells. Human Cytochrome c present in the sample or calibrator binds to antibodies adsorbed to the microwells. A biotin-conjugated anti- human Cytochrome c antibody is added and binds to human Cytochrome c captured by the first antibody. Following incubation unbound biotin-conjugated anti-human Cytochrome c antibody is removed during a wash step. Streptavidin-HRP is added and binds to the biotin-conjugated anti- human Cytochrome c antibody. Following incubation unbound Streptavidin-HRP is removed during a wash step, and substrate solution reactive with HRP is added to the wells. A colored product is formed in proportion to the amount of human Cytochrome c present in the sample or calibrator. The reaction is terminated by addition of acid and absorbance is measured at 450 nm. A calibration curve is prepared from 7 human Cytochrome c calibrator dilutions and human Cytochrome c sample concentration determined.

Components: 1 aluminum pouch with a Microwell Plate coated with monoclonal antibody to human Cytochrome c
1 vial (100 µL) Biotin-Conjugate anti-human Cytochrome c monoclonal antibody
1 vial (150 µL) Streptavidin-HRP
2 vials human Cytochrome c Calibrator lyophilized, 10 ng/mL upon reconstitution
1 bottle (50 mL) Wash Buffer Concentrate 20X (PBS with 1% Tween 20)
1 vial (5 mL) Assay Buffer Concentrate 20X (PBS with 1% Tween 20 and protein stabilizer)
1 bottle (15 mL) Lysis Buffer 10X
1 vial (15 mL) Substrate Solution (tetramethyl-benzidine)
1 vial (15 mL) Stop Solution (1M Phosphoric acid)
2 vials (0.4 mL each), Green Dye, Red Dye
4 adhesive Plate Covers.

Material not included: 5 mL and 10 mL graduated pipettes
5 µL to 1,000 µL adjustable single channel micropipettes with disposable tips
50 µL to 300 µL adjustable multichannel micropipette with disposable tips
Multichannel micropipette reservoir
Beakers, flasks, cylinders necessary for preparation of reagents
Device for delivery of wash solution (multichannel wash bottle or automatic wash system)
Microwell strip reader capable of reading at 450 nm (620 nm as optional reference wave length)
Glass-distilled or de-ionized water
Statistical calculator with program to perform regression analysis

Application Details

Plate: Pre-coated

Reagent Preparation:

Buffer Concentrates should be brought to room temperature and should be diluted before starting the test procedure. If crystals have formed in the Buffer Concentrates, warm them gently until they have completely dissolved.

A. Wash Buffer (1X): Pour entire contents (50 mL) of the Wash Buffer Concentrate (20X) into a clean 1,000 mL graduated cylinder. Bring to final volume of 1,000 mL with glass-distilled or de-ionized water. Mix gently to avoid foaming. Transfer to a clean wash bottle and store at 2°to 25°C. Please note that the Wash Buffer (1X) is stable for 30 days.

B. Assay Buffer (1X): Pour the entire contents (5 mL) of the Assay Buffer Concentrate (20X) into a clean 100 mL graduated cylinder. Bring to final volume of 100 mL with distilled water. Mix gently to avoid foaming. Store at 4°C. Please note that the Assay Buffer (1X) is stable for 30 days.

C. Preparation of Biotin-Conjugate: Please note that the Biotin-Conjugate should be used within 30 minutes after dilution. Make a 1:100 dilution of the concentrated Biotin-Conjugate solution with Assay Buffer (1X) in a clean plastic tube as needed.

D. Preparation of Human Cytochrome c Calibrator: Reconstitute human Cytochrome c Calibrator by addition of distilled water. Reconstitution volume is stated on the label of the calibrator vial. Swirl or mix gently to insure complete and homogeneous solubilization (concentration of reconstituted calibrator = 10 ng/mL). Allow the calibrator to reconstitute for 10-30 minutes. Mix well prior to making dilutions. After usage remaining calibrator cannot be stored and has to be discarded.

E. Preparation of Streptavidin-HRP: Please note that the Streptavidin-HRP should be used within 30 minutes after dilution. Make a 1:200 dilution of the concentrated Streptavidin-HRP solution with Assay Buffer (1X) in a clean plastic tube as needed

F. Preparation of Lysis Buffer: Pour the entire contents (15 mL) of the Lysis Buffer Concentrate (10X) into a clean 150 mL graduated cylinder. Bring to final volume of 150 mL with distilled or de-ionized water and mix gently. Store at room temperature. Please note that the Lysis Buffer is stable for 30 days.

G. Addition of color-giving reagents: Green Dye, Red Dye: In order to help our customers to avoid any mistakes in pipetting the Human Cytochrome c ELISA, we offer a tool that helps to monitor the addition of even very small volumes of a solution to the reaction well by giving distinctive colors to each step of the ELISA procedure. This procedure is optional, does not in any way interfere with the test results, and is designed to help the customer with the performance of the test, but can also be omitted, just following the package insert.

Calculation of Results:

Calculate the mean absorbance values for each set of duplicate calibrators and samples. Duplicates should be within 20 per cent of the mean value.
Create a calibration curve by plotting the mean absorbance for each calibrator concentration on the ordinate against the human Cytochrome c concentration on the abscissa. Draw a best fit curve through the points of the graph (a 5- parameter curve fit is recommended).
To determine the concentration of circulating human Cytochrome c for each sample, first find the mean absorbance value on the ordinate and extend a horizontal line to the calibration curve. At the point of intersection, extend a vertical line to the abscissa and read the corresponding human Cytochrome c concentration.
For samples which have been diluted according to the instructions given in this protocol, the concentration read from the calibration curve must be multiplied by the dilution factor.
Calculation of samples with a concentration exceeding calibrator 1 may result in incorrect, low human Cytochrome c levels. Such samples require further external predilution according to expected human Cytochrome c values with Assay Buffer (1X) in order to precisely quantitate the actual human Cytochrome c level.
It is suggested that each testing facility establishes a control sample of known human Cytochrome c concentration and runs this additional control with each assay. If the values obtained are not within the expected range of the control, the assay results may be invalid.

Restrictions: For Research Use only

Handling

Storage: 4 °C

Storage Comment: Store kit reagents at 4°C. Immediately after use remaining reagents should be returned to cold storage (4°C). Expiration date of the kit and reagents is stated on labels. The expiration date of the kit components can only be guaranteed if the components are stored properly, and if, in case of repeated use of one component, this reagent is not contaminated by the first handling.

Expiry Date: The expiry date is stated on the label.

Target Details for Cytochrome C

Target: Cytochrome C (CYCS) (Cytochrome C, Somatic (CYCS))

Alternative Name: Cytochrome C

Background: Apoptotic cell death is a fundamental feature of virtually all cells. It is an indispensable process during normal development, tissue homeostasis, development of the nervous system and the regulation of the immune system. Insufficient or excessive cell death can contribute to human disease, including cancer or degenerative disorders. The highly coordinated and stereotyped manner of this induced cell death suggests that the cells activate a common death program, towards which diverse signal-transducing pathways converge. The mitochondria turned out to participate in the central control or executioner phase of the cell death cascade. Cytochrome c was identified as a component required for the crucial steps in apoptosis, caspase-3 activation and DNA fragmentation. Cytochrome c was shown to redistribute from mitochondria to cytosol during apoptosis in intact cells. Mitochondrial cytochrome c is a water-soluble protein of 15 kDa with a net positive charge, residing loosely attached in the mitochondrial intermembrane space. Cytochrome c functions in the respiratory chain by interaction with redox partners. It is highly conserved during evolution. Like most mitochondrial proteins cytochrome c is encoded by a nuclear gene and synthesized as a cytoplasmic precursor molecule, apocytochrome c, which becomes selectively imported into the mitochondrial intermembrane space. The molecular mechanisms responsible for the translocation of cytochrome c from mitochondria to cytosol during apoptosis are unknown. A reduction in mitochondrial transmembrane potential has been reported to accompany early apoptosis. The release of cytochrome c into the cytosol leads to an activation of an apoptotic program via activation of a caspase dependent pathway. Cytochrome c achieves this goal by interaction with other cytosolic factors forming a complex (apoptosome) composed of cytochrome c, Apaf-1, dATP and Apaf-3/caspase 9. Bcl-2 on the other hand was shown to be able to prevent apoptosis by blocking the release of cytochrome c from mitochondria. Measurement of cytochrome c release from the mitochondria is a tool to detect the first early steps for initiating apoptosis in cells. Cytochrome c release in the cytosol occurs prior to the activation of caspases and DNA fragmentation which is considered the hallmark of apoptosis. Detection of cytochrome c released from the mitochondria to the cytoplasm can be achieved by a selective lysis of the cell membrane. Very recently it has been shown that this mitochondria dwelling molecule can be detected in the medium already 1 hour after apoptosis. Moreover, elevated cytochrome c levels were observed in serum from patients with hematological malignancies. In the course of cancer chemotherapy, the serum cytochrome c level grew rapidly and it decreased gradually as the patient was cleared from malignant cells. Thus, serum cytochrome c monitoring might serve as a marker indicating the onset of apoptosis and cell turnover in vivo.

Pathways: Apoptosis, Caspase Cascade in Apoptosis, Positive Regulation of Endopeptidase Activity