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Cytochrome c Colorimetric Cell-Based ELISA Kit

SKU:
CBCAB00616
Product Type:
ELISA Kit
ELISA Type:
Cell Based
Research Area:
Cell Death
Reactivity:
Human
Mouse
Rat
Detection Method:
Colorimetric
€599
Frequently bought together:

Description

system_update_altDatasheetMSDS

Cytochrome c Colorimetric Cell-Based ELISA Kit

The Cytochrome C Colorimetric Cell-Based ELISA Kit from Assay Genie is a cutting-edge tool designed for the accurate detection of cytochrome C levels in cell lysates and tissue homogenates. This kit offers high sensitivity and specificity, allowing for precise and reproducible results in a variety of research applications.Cytochrome C is a vital protein located in the mitochondria and plays a crucial role in the process of apoptosis, or programmed cell death. Dysregulation of cytochrome C levels has been linked to various diseases, including cancer, Alzheimer's, and Parkinson's disease, making it a valuable biomarker for disease research and therapeutic development.

With the Cytochrome C Colorimetric Cell-Based ELISA Kit, researchers can confidently study the role of cytochrome C in cellular processes and disease pathogenesis, paving the way for innovative treatments and diagnostic strategies. Trust Assay Genie for reliable and accurate results in your research endeavors.

Product Name:Cytochrome c Colorimetric Cell-Based ELISA
Product Code:CBCAB00616
ELISA Type:Cell-Based
Target:Cytochrome c
Reactivity:Human, Mouse, Rat
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:96-Well Microplate

The Cytochrome c Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect Cytochrome c protein expression profile in cells. The kit can be used for measuring the relative amounts of Cytochrome c in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on Cytochrome c.

Qualitative determination of Cytochrome c concentration is achieved by an indirect ELISA format. In essence, Cytochrome c is captured by Cytochrome c-specific primary antibodies while the HRP-conjugated secondary antibodies bind the Fc region of the primary antibody. Through this binding, the HRP enzyme conjugated to the secondary antibody can catalyze a colorimetric reaction upon substrate addition. Due to the qualitative nature of the Cell-Based ELISA, multiple normalization methods are needed:

1. A monoclonal antibody specific for human GAPDH is included to serve as an internal positive control in normalizing the target absorbance values.
2. Following the colorimetric measurement of HRP activity via substrate addition, the Crystal Violet whole-cell staining method may be used to determine cell density. After staining, the results can be analysed by normalizing the absorbance values to cell amounts, by which the plating difference can be adjusted.
Database Information:Gene ID: 54205, UniProt ID: P99999, OMIM: 123970, Unigene: Hs.437060/Hs.617193
Gene Symbol:CYCS
Sub Type:None
UniProt Protein Function:CYCS: Electron carrier protein. The oxidized form of the cytochrome c heme group can accept an electron from the heme group of the cytochrome c1 subunit of cytochrome reductase. Cytochrome c then transfers this electron to the cytochrome oxidase complex, the final protein carrier in the mitochondrial electron-transport chain. Belongs to the cytochrome c family.
UniProt Protein Details:

Protein type:Apoptosis; Oxidoreductase; Mitochondrial; Protein phosphatase, Ser/Thr (non-receptor)

Chromosomal Location of Human Ortholog: 7p15.3

Cellular Component: cytosol; mitochondrial inner membrane; mitochondrial intermembrane space; mitochondrion; nucleus; protein phosphatase type 2A complex

Molecular Function:electron transporter, transferring electrons from CoQH2-cytochrome c reductase complex and cytochrome c oxidase complex activity; heme binding; protein binding; protein serine/threonine phosphatase activity

Biological Process: caspase activation via cytochrome c; cellular respiration; mitochondrial electron transport, cytochrome c to oxygen; mitochondrial electron transport, ubiquinol to cytochrome c; mitochondrion organization and biogenesis; response to reactive oxygen species

Disease: Thrombocytopenia 4

NCBI Summary:This gene encodes a small heme protein that functions as a central component of the electron transport chain in mitochondria. The encoded protein associates with the inner membrane of the mitochondrion where it accepts electrons from cytochrome b and transfers them to the cytochrome oxidase complex. This protein is also involved in initiation of apoptosis. Mutations in this gene are associated with autosomal dominant nonsyndromic thrombocytopenia. Numerous processed pseudogenes of this gene are found throughout the human genome.[provided by RefSeq, Jul 2010]
UniProt Code:P99999
NCBI GenInfo Identifier:42560196
NCBI Gene ID:54205
NCBI Accession:P99999.2
UniProt Secondary Accession:P99999,P00001, Q6NUR2, Q6NX69, Q96BV4, A4D166, B2R4I1
UniProt Related Accession:P99999
Molecular Weight:11,749 Da
NCBI Full Name:Cytochrome c
NCBI Synonym Full Names:cytochrome c, somatic
NCBI Official Symbol:CYCS  
NCBI Official Synonym Symbols:CYC; HCS; THC4  
NCBI Protein Information:cytochrome c
UniProt Protein Name:Cytochrome c
Protein Family:Cytochrome
UniProt Gene Name:CYCS  
UniProt Entry Name:CYC_HUMAN
ComponentQuantity
96-Well Cell Culture Clear-Bottom Microplate2 plates
10X TBS24 mL
Quenching Buffer24 mL
Blocking Buffer50 mL
15X Wash Buffer50 mL
Primary Antibody Diluent12 mL
100x Anti-Phospho Target Antibody 60 µL
100x Anti-Target Antibody60 µL
Anti-GAPDH Antibody60 µL
HRP-Conjugated Anti-Rabbit IgG Antibody12 mL
HRP-Conjugated Anti-Mouse IgG Antibody12 mL
SDS Solution12 mL
Stop Solution24 mL
Ready-to-Use Substrate12 mL
Crystal Violet Solution12 mL
Adhesive Plate Seals2 seals

The following materials and/or equipment are NOT provided in this kit but are necessary to successfully conduct the experiment:

  • Microplate reader able to measure absorbance at 450 nm and/or 595 nm for Crystal Violet Cell Staining (Optional)
  • Micropipettes with capability of measuring volumes ranging from 1 µL to 1 ml
  • 37% formaldehyde (Sigma Cat# F-8775) or formaldehyde from other sources
  • Squirt bottle, manifold dispenser, multichannel pipette reservoir or automated microplate washer
  • Graph paper or computer software capable of generating or displaying logarithmic functions
  • Absorbent papers or vacuum aspirator
  • Test tubes or microfuge tubes capable of storing ≥1 ml
  • Poly-L-Lysine (Sigma Cat# P4832 for suspension cells)
  • Orbital shaker (optional)
  • Deionized or sterile water

*Note: Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.

StepProcedure
1.Seed 200 µL of 20,000 adherent cells in culture medium in each well of a 96-well plate. The plates included in the kit are sterile and treated for cell culture. For suspension cells and loosely attached cells, coat the plates with 100 µL of 10 µg/ml Poly-L-Lysine (not included) to each well of a 96-well plate for 30 minutes at 37°C prior to adding cells.
2.Incubate the cells for overnight at 37°C, 5% CO2.
3.Treat the cells as desired.
4.Remove the cell culture medium and rinse with 200 µL of 1x TBS, twice.
5.Fix the cells by incubating with 100 µL of Fixing Solution for 20 minutes at room temperature. The 4% formaldehyde is used for adherent cells and 8% formaldehyde is used for suspension cells and loosely attached cells.
6.Remove the Fixing Solution and wash the plate 3 times with 200 µL 1x Wash Buffer for five minutes each time with gentle shaking on the orbital shaker. The plate can be stored at 4°C for a week.
7.Add 100 µL of Quenching Buffer and incubate for 20 minutes at room temperature.
8.Wash the plate 3 times with 1x Wash Buffer for 5 minutes each time.
9.Add 200 µL of Blocking Buffer and incubate for 1 hour at room temperature.
10. Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time.
11.Add 50 µL of 1x primary antibodies (Anti-Cytochrome c Antibody and/or Anti-GAPDH Antibody) to the corresponding wells, cover with Parafilm and incubate for 16 hours (overnight) at 4°C. If the target expression is known to be high, incubate for 2 hours at room temperature.
12.Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time.
13.Add 50 µL of 1x secondary antibodies (HRP-Conjugated AntiRabbit IgG Antibody or HRP-Conjugated Anti-Mouse IgG Antibody) to corresponding wells and incubate for 1.5 hours at room temperature.
14.Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time.
15.Add 50 µL of Ready-to-Use Substrate to each well and incubate for 30 minutes at room temperature in the dark.
16.Add 50 µL of Stop Solution to each well and read OD at 450 nm immediately using the microplate reader.

(Additional Crystal Violet staining may be performed if desired – details of this may be found in the kit technical manual.)

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