Mouse UCP-1 (Uncoupling Protein 1, Mitochondrial) CLIA Kit (MOES00400)

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SKU:
MOES00400
€649

Description

ELISA Kit Technical ManualMSDS

Mouse UCP-1 (Uncoupling Protein 1, Mitochondrial) 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 UCP-1 . 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 UCP-1 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 UCP-1, 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 UCP-1. The concentration of Mouse UCP-1 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 range31.25-2000 pg/mL
Sensitivity18.75 pg/mL
Sample volume100uL
Sample typeTissue homogenates
RepeatabilityCV < 15%

Specificity

This kit recognizes Mouse UCP-1 in samples. No significant cross-reactivity or interference between Mouse UCP-1 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
(pg/mL)
RLU Average Corrected
200054280
57162
5572155693
100023843
24105
2397423946
50011299
10765
1103211004
2505065
5523
52945266
1252701
2515
26082580
62.51385
1237
13111283
31.25644
704
674646
028
28
28--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Mouse UCP-1 were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Mouse UCP-1 were tested on 3 different plates, 20 replicates in each plate.

  Intra-assay Precision Inter-assay Precision
Sample123123
n202020202020
Mean (pg/mL)98.08198.00694.5193.34194.03644.60
Standard deviation9.0823.5448.9611.1220.3343.25
C V (%)9.2611.897.0511.9110.486.71

Recovery

The recovery of Mouse UCP-1 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)100-115108
EDTA plasma (n=5)97-113105
Cell culture media (n=5)88-10495

Linearity

Samples were spiked with high concentrations of Mouse UCP-1 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 (%)100-11991-10899-117
Average (%)10998107
1:4Range (%)98-109102-11998-110
Average (%)104108103
1:8Range (%)85-9898-11794-108
Average (%)9110799
1:16Range (%)102-11585-10195-112
Average (%)10892102

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 UCP-1 (Uncoupling Protein 1, Mitochondrial) CLIA Kit (MOES00400) 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 UCP-1 (Uncoupling Protein 1, Mitochondrial) CLIA Kit (MOES00400) Protein Information

UniProt Protein Function:UCP1: UCP are mitochondrial transporter proteins that create proton leaks across the inner mitochondrial membrane, thus uncoupling oxidative phosphorylation from ATP synthesis. As a result, energy is dissipated in the form of heat. Belongs to the mitochondrial carrier family.
UniProt Protein Details:

Protein type:Membrane protein, integral; Membrane protein, multi-pass; Mitochondrial

Chromosomal Location of Human Ortholog: 8 C2|8 39.65 cM

Cellular Component: integral component of membrane; membrane; mitochondrial envelope; mitochondrial inner membrane; mitochondrial membrane; mitochondrion

Molecular Function:GDP binding; GTP binding; oxidative phosphorylation uncoupler activity; protein binding; purine ribonucleotide binding; transmembrane transporter activity

Biological Process: brown fat cell differentiation; cellular response to hormone stimulus; diet induced thermogenesis; ion transport; mitochondrial transport; proton transport; regulation of transcription from RNA polymerase II promoter; response to cold; response to nutrient levels; response to temperature stimulus; transport

UniProt Code:P12242
NCBI GenInfo Identifier:6678497
NCBI Gene ID:22227
NCBI Accession:NP_033489.1
UniProt Related Accession:P12242
Molecular Weight:
NCBI Full Name:mitochondrial brown fat uncoupling protein 1
NCBI Synonym Full Names:uncoupling protein 1 (mitochondrial, proton carrier)
NCBI Official Symbol:Ucp1  
NCBI Official Synonym Symbols:Ucp; Slc25a7; AI385626  
NCBI Protein Information:mitochondrial brown fat uncoupling protein 1
UniProt Protein Name:Mitochondrial brown fat uncoupling protein 1
UniProt Synonym Protein Names:Solute carrier family 25 member 7
Protein Family:Mitochondrial brown fat uncoupling protein
UniProt Gene Name:Ucp1  
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Additional Information

Reactivity:
Mouse
ELISA Type:
CLIA
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