Rat Cyt-C (Cytochrome C) CLIA Kit (RTES00006)

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ELISA Kit Technical ManualMSDS

Rat Cyt-C (Cytochrome C) CLIA Kit

This kit uses Sandwich-CLIA as the method. The micro CLIA plate provided in this kit has been pre-coated with an antibody specific to Rat Cyt-C . Standards or samples are added to the appropriate micro CLIA plate wells and combined with the specific antibody. Then a biotinylated detection antibody specific for Rat Cyt-C and Avidin-Horseradish Peroxidase (HRP) conjugate are added to each micro plate well successively and incubated. Free components are washed away. The substrate solution is added to each well. Only those wells that contain Rat Cyt-C, biotinylated detection antibody and Avidin-HRP conjugate will appear fluorescence. The Relative light unit (RLU) value is measured spectrophotometrically by the Chemiluminescence immunoassay analyzer. The RLU value is positively associated with the concentration of Rat Cyt-C. The concentration of Rat Cyt-C in the samples can be calculated by comparing the RLU of the samples to the standard curve.

Assay typeSandwich
Assay time4.5h
Detection methodChemiluminescence
Detection range78.13-5000 pg/mL
Sensitivity46.88 pg/mL
Sample volume100uL
Sample typeSerum, plasma and other biological fluids
RepeatabilityCV < 15%


This kit recognizes Rat Cyt-C in samples. No significant cross-reactivity or interference between Rat Cyt-C and analogues was observed.

Typical Data

As the RLU values of the standard curve may vary according to the conditions of the actual assay performance (e.g. operator, pipetting technique, washing technique or temperature effects), the operator should establish a standard curve for each test. Typical standard curve and data is provided below for reference only.

RLU Average Corrected


Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Rat Cyt-C were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Rat Cyt-C were tested on 3 different plates, 20 replicates in each plate.

  Intra-assay Precision Inter-assay Precision
Mean (pg/mL)257.49689.881774.80266.14739.711631.97
Standard deviation24.3870.85141.6332.3470.57141.17
C V (%)9.4710.277.9812.159.548.65


The recovery of Rat Cyt-C spiked at three different levels in samples throughout the range of the assay was evaluated in various matrices.

Sample Type Range (%) Average Recovery (%)
Serum (n=5)93-10898
EDTA plasma (n=5)87-9992
Cell culture media (n=5)88-10596


Samples were spiked with high concentrations of Rat Cyt-C and diluted with Reference Standard & Sample Diluent to produce samples with values within the range of the assay.

    Serum (n=5) EDTA plasma (n=5) Cell culture media (n=5)
1:2Range (%)104-11799-11490-105
Average (%)11010796
1:4Range (%)89-10498-11291-103
Average (%)9510396
1:8Range (%)102-11895-112101-113
Average (%)108102107
1:16Range (%)101-11592-105102-116
Average (%)10998108

Kit Components & Storage

An unopened kit can be stored at 4'C for 1 month. If the kit is not used within 1 month, store the items separately according to the following conditions once the kit is received.

Item Specifications Storage
Micro CLIA Plate(Dismountable)8 wells X12 strips-20'C, 6 months
Reference Standard2 vials
Concentrated Biotinylated Detection Ab (100X)1 vial, 120 uL
Concentrated HRP Conjugate (100X)1 vial, 120 uL-20'C(shading light), 6 months
Reference Standard & Sample Diluent1 vial, 20 mL4'C, 6 months
Biotinylated Detection Ab Diluent1 vial, 14 mL
HRP Conjugate Diluent1 vial, 14 mL
Concentrated Wash Buffer (25X)1 vial, 30 mL
Substrate Reagent A1 vial, 5 mL4'C (shading light)
Substrate Reagent B1 vial, 5 mL4'C (shading light)
Plate Sealer5 pieces
Product Description1 copy
Certificate of Analysis1 copy

Rat Cyt-C (Cytochrome C) CLIA Kit (RTES00006) Assay procedure

    1. Set standard, test sample and control (zero) wells on the pre-coated plate and record theirpositions. It is recommended to measure each standard and sample in duplicate. Note: addall solutions to the bottom of the plate wells while avoiding contact with the well walls. Ensuresolutions do not foam when adding to the wells.
    2. Aliquot 100µl of standard solutions into the standard wells.
    3. Add 100µl of Sample / Standard dilution buffer into the control (zero) well.
    4. Add 100µl of properly diluted sample (serum, plasma, tissue homogenates and otherbiological fluids.) into test sample wells.
    5. Cover the plate with the sealer provided in the kit and incubate for 90 min at 37°C.
    6. Aspirate the liquid from each well, do not wash. Immediately add 100µL of BiotinylatedDetection Ab working solution to each well. Cover the plate with a plate seal and gently mix.Incubate for 1 hour at 37°C.
    7. Aspirate or decant the solution from the plate and add 350µL of wash buffer to each welland incubate for 1-2 minutes at room temperature. Aspirate the solution from each well andclap the plate on absorbent filter paper to dry. Repeat this process 3 times. Note: a microplatewasher can be used in this step and other wash steps.
    8. Add 100µL of HRP Conjugate working solution to each well. Cover with a plate seal andincubate for 30 min at 37°C.
    9. Aspirate or decant the solution from each well. Repeat the wash process for five times asconducted in step 7.
    10. Add 100µL of Substrate mixture solution to each well. Cover with a new plate seal andincubate for no more than 5 min at 37°C. Protect the plate from light.
    11. Determine the RLU value of each well immediately.

Rat Cyt-C (Cytochrome C) CLIA Kit (RTES00006) Protein Information

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

Cellular Component: cytosol; mitochondrial inner membrane; mitochondrial intermembrane space; mitochondrial respiratory chain; mitochondrion; nucleus

Molecular Function:electron carrier activity; electron transporter, transferring electrons from CoQH2-cytochrome c reductase complex and cytochrome c oxidase complex activity; heme binding; lipid binding; metal ion binding

Biological Process: apoptosis; caspase activation via cytochrome c; hydrogen peroxide metabolic process; mitochondrial electron transport, cytochrome c to oxygen; mitochondrial electron transport, ubiquinol to cytochrome c; response to oxidative stress

UniProt Code:P62898
NCBI GenInfo Identifier:51702227
NCBI Gene ID:100363502
NCBI Accession:P62898.2
UniProt Secondary Accession:P62898,P00009, Q8C2S2,
Molecular Weight:11,605 Da
NCBI Full Name:Cytochrome c, somatic
NCBI Synonym Full Names:cytochrome c, somatic-like
NCBI Official Symbol:LOC100363502
NCBI Protein Information:cytochrome c, somatic-like
UniProt Protein Name:Cytochrome c, somatic
Protein Family:Cytochrome
UniProt Gene Name:Cycs
UniProt Entry Name:CYC_RAT
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