Rat NFIL3 CLIA Kit (RTES00414)

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RTES00414
€649

Description

ELISA Kit Technical ManualMSDS

Rat NFIL3 (Nuclear Factor, Interleukin 3 Regulated) 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 NFIL3 . 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 NFIL3 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 NFIL3, 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 NFIL3. The concentration of Rat NFIL3 in the samples can be calculated by comparing the RLU of the samples to the standard curve.

Assay typeSandwich
Format96T
Assay time4.5h
ReactivityRat
Detection methodChemiluminescence
Detection range31.25-2000 pg/mL
Sensitivity18.75 pg/mL
Sample volume100uL
Sample typeSerum, plasma and other biological fluids
RepeatabilityCV < 15%

Specificity

This kit recognizes Rat NFIL3 CLIA Kit in samples. No significant cross-reactivity or interference between Rat NFIL3 CLIA Kit 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
200054480
56962
5572155693
100023603
24345
2397423946
50011094
10970
1103211004
2505044
5544
52945266
1252798
2418
26082580
62.51378
1244
13111283
31.25666
682
674646
027
29
28--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Rat NFIL3 CLIA Kit were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Rat NFIL3 CLIA Kit were tested on 3 different plates, 20 replicates in each plate.

  Intra-assay Precision Inter-assay Precision
Sample123123
n202020202020
Mean (pg/mL)108.47258.99984.87108.97278.11992.46
Standard deviation13.2727.19104.4912.3528.1286.34
C V (%)12.2310.5010.6111.3310.118.70

Recovery

The recovery of Rat NFIL3 CLIA Kit 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)92-10999
EDTA plasma (n=5)86-9993
Cell culture media (n=5)89-10596

Linearity

Samples were spiked with high concentrations of Rat NFIL3 CLIA Kit 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 (%)102-11993-10999-115
Average (%)110101105
1:4Range (%)90-104102-11793-106
Average (%)98108100
1:8Range (%)101-11598-10996-109
Average (%)107103102
1:16Range (%)102-12094-10786-101
Average (%)10910093

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 NFIL3 CLIA Kit (RTES00414) 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 NFIL3 CLIA Kit (RTES00414) Protein Information

UniProt Protein Function:E4BP4: Acts as a transcriptional regulator that recognizes and binds to the sequence 5'-[GA]TTA[CT]GTAA[CT]-3', a sequence present in many cellular and viral promoters. Represses transcription from promoters with activating transcription factor (ATF) sites. Represses promoter activity in osteoblasts. Represses transcriptional activity of PER1. Represses transcriptional activity of PER2 via the B- site on the promoter. Activates transcription from the interleukin-3 promoter in T-cells. Competes for the same consensus-binding site with PAR DNA-binding factors (DBP, HLF and TEF). Component of the circadian clock that acts as a negative regulator for the circadian expression of PER2 oscillation in the cell-autonomous core clock. Protects pro-B cells from programmed cell death. Belongs to the bZIP family. NFIL3 subfamily.
UniProt Protein Details:

Protein type:DNA-binding; Transcription factor

Cellular Component: nucleus

Molecular Function:transcription factor activity

Biological Process: transcription from RNA polymerase II promoter; circadian rhythm; immune response; negative regulation of transcription from RNA polymerase II promoter

NCBI Summary:The protein encoded by this gene is a transcriptional regulator that binds as a homodimer to activating transcription factor (ATF) sites in many cellular and viral promoters. The encoded protein represses Per1 and Per2 expression and therefore plays a role in the regulation of circadian rhythm. [provided by RefSeq, Feb 2014]
UniProt Code:Q6IMZ0
NCBI GenInfo Identifier:48675850
NCBI Gene ID:114519
NCBI Accession:NP_446179.2
UniProt Secondary Accession:Q6IMZ0,Q923M2,
UniProt Related Accession:Q6IMZ0
Molecular Weight:50,781 Da
NCBI Full Name:nuclear factor interleukin-3-regulated protein
NCBI Synonym Full Names:nuclear factor, interleukin 3 regulated
NCBI Official Symbol:Nfil3  
NCBI Official Synonym Symbols:E4BP4  
NCBI Protein Information:nuclear factor interleukin-3-regulated protein
UniProt Protein Name:Nuclear factor interleukin-3-regulated protein
UniProt Synonym Protein Names:E4 promoter-binding protein 4
Protein Family:Nuclear factor interleukin-3-regulated protein
UniProt Gene Name:Nfil3  
UniProt Entry Name:NFIL3_RAT
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Additional Information

Reactivity:
Rat
ELISA Type:
CLIA
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