Mouse M2-PK (Pyruvate Kinase) CLIA Kit (MOES00514)

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MOES00514
€649

Description

ELISA Kit Technical ManualMSDS

Mouse M2-PK (Pyruvate Kinase) 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 Mouse M2-PK . 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 Mouse M2-PK 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 Mouse M2-PK, 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 Mouse M2-PK. The concentration of Mouse M2-PK in the samples can be calculated by comparing the RLU of the samples to the standard curve.

Assay typeSandwich
Format96T
Assay time4.5h
ReactivityMouse
Detection methodChemiluminescence
Detection range0.16-10 IU/mL
Sensitivity0.09 IU/mL
Sample volume100uL
Sample typeSerum, plasma and other biological fluids
RepeatabilityCV < 15%

Specificity

This kit recognizes Mouse M2-PK in samples. No significant cross-reactivity or interference between Mouse M2-PK 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.

Concentration
(IU/mL)
RLU Average Corrected
1032044
32898
3247132447
514169
16771
1547015446
2.57737
7269
75037479
1.253370
3936
36533629
0.631778
1744
17611737
0.31844
804
824800
0.16343
371
357333
023
25
24--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Mouse M2-PK were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Mouse M2-PK were tested on 3 different plates, 20 replicates in each plate.

  Intra-assay Precision Inter-assay Precision
Sample123123
n202020202020
Mean (IU/mL)0.541.524.310.501.484.03
Standard deviation0.050.140.490.050.120.28
C V (%)9.269.2111.3710.008.116.95

Recovery

The recovery of Mouse M2-PK 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)97-109103
EDTA plasma (n=5)88-10294
Cell culture media (n=5)86-10093

Linearity

Samples were spiked with high concentrations of Mouse M2-PK 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 (%)86-100101-116103-114
Average (%)92110108
1:4Range (%)85-10090-10092-109
Average (%)9195100
1:8Range (%)96-111101-11588-101
Average (%)10410794
1:16Range (%)93-10693-10890-104
Average (%)10110195

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

Mouse M2-PK (Pyruvate Kinase) CLIA Kit (MOES00514) 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.

Mouse M2-PK (Pyruvate Kinase) CLIA Kit (MOES00514) Protein Information

UniProt Protein Function:Glycolytic enzyme that catalyzes the transfer of a phosphoryl group from phosphoenolpyruvate (PEP) to ADP, generating ATP. Stimulates POU5F1-mediated transcriptional activation.
UniProt Code:P52480
NCBI GenInfo Identifier:146345448
NCBI Gene ID:18746
NCBI Accession:P52480.4
UniProt Secondary Accession:P52480,Q3TBV8, Q3TBW5, Q3TC59, Q3U1X3, Q3U5P6, Q4VC20 Q64484, Q91YI8, Q9CWB1,
UniProt Related Accession:P52480
Molecular Weight:57,985 Da
NCBI Full Name:Pyruvate kinase PKM
NCBI Synonym Full Names:pyruvate kinase, muscle
NCBI Official Symbol:Pkm  
NCBI Official Synonym Symbols:Pk3; Pk-2; Pk-3; Pkm2; AA414905; AL024370; AL024424  
NCBI Protein Information:pyruvate kinase PKM
UniProt Protein Name:Pyruvate kinase PKM
UniProt Synonym Protein Names:Pyruvate kinase muscle isozyme
UniProt Gene Name:Pkm  
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Additional Information

Reactivity:
Mouse
ELISA Type:
CLIA
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