Rat GR (Glucocorticoid Receptor) CLIA Kit (RTES00239)

SKU:
RTES00239
€649

Description

ELISA Kit Technical ManualMSDS

Rat GR (Glucocorticoid Receptor) 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 GR . 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 GR 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 GR, 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 GR. The concentration of Rat GR 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 GR in samples. No significant cross-reactivity or interference between Rat GR 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
200051986
59456
5572155693
100022274
25674
2397423946
50011656
10408
1103211004
2505049
5539
52945266
1252724
2492
26082580
62.51390
1232
13111283
31.25648
700
674646
027
29
28--

Precision

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

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

  Intra-assay Precision Inter-assay Precision
Sample123123
n202020202020
Mean (pg/mL)98.91196.48690.66106.70200.99735.86
Standard deviation9.0620.9677.8410.2615.7275.13
C V (%)9.1610.6711.279.627.8210.21

Recovery

The recovery of Rat GR 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)91-10596
EDTA plasma (n=5)86-9892
Cell culture media (n=5)88-10193

Linearity

Samples were spiked with high concentrations of Rat GR 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 (%)92-10891-10799-116
Average (%)9997106
1:4Range (%)91-10689-10389-103
Average (%)999497
1:8Range (%)91-10499-11491-103
Average (%)9810498
1:16Range (%)95-10698-11187-98
Average (%)10010593

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 GR (Glucocorticoid Receptor) CLIA Kit (RTES00239) 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 GR (Glucocorticoid Receptor) CLIA Kit (RTES00239) Protein Information

UniProt Protein Function:GR: transcription factor of the nuclear receptor family. Receptor for glucocorticoids (GC). Binds to glucocorticoid response elements (GRE) and modulates other transcription factors. Affects inflammatory responses, cellular proliferation and differentiation in target tissues. Three alternatively spliced isoforms have been described.
UniProt Protein Details:

Protein type:Transcription factor; DNA-binding; Mitochondrial; Nuclear receptor

Chromosomal Location of Human Ortholog: 5q31.3

Cellular Component: nucleoplasm; membrane; mitochondrial matrix; cytoplasm; cytosol; nucleus

Molecular Function:protein dimerization activity; glucocorticoid receptor activity; protein binding; zinc ion binding; transcription factor activity; steroid binding

Biological Process: transcription from RNA polymerase II promoter; transcription initiation from RNA polymerase II promoter; transcription, DNA-dependent; maternal behavior; adrenal gland development; regulation of glucocorticoid biosynthetic process; chromatin modification; signal transduction; glucocorticoid metabolic process; glucocorticoid mediated signaling; regulation of transcription, DNA-dependent; positive regulation of neuron apoptosis; glucocorticoid receptor signaling pathway; gene expression; positive regulation of transcription from RNA polymerase II promoter; regulation of gluconeogenesis

Disease: Glucocorticoid Resistance, Generalized

NCBI Summary:This gene encodes glucocorticoid receptor, which can function both as a transcription factor that binds to glucocorticoid response elements in the promoters of glucocorticoid responsive genes to activate their transcription, and as a regulator of other transcription factors. This receptor is typically found in the cytoplasm, but upon ligand binding, is transported into the nucleus. It is involved in inflammatory responses, cellular proliferation, and differentiation in target tissues. Mutations in this gene are associated with generalized glucocorticoid resistance. Alternative splicing of this gene results in transcript variants encoding either the same or different isoforms. Additional isoforms resulting from the use of alternate in-frame translation initiation sites have also been described, and shown to be functional, displaying diverse cytoplasm-to-nucleus trafficking patterns and distinct transcriptional activities (PMID:15866175). [provided by RefSeq, Feb 2011]
UniProt Code:P06536
NCBI GenInfo Identifier:4504133
NCBI Gene ID:2908
NCBI Accession:NP_000167.1
UniProt Secondary Accession:P06536,P06537, P06536,
UniProt Related Accession:P04150
Molecular Weight:
NCBI Full Name:glucocorticoid receptor isoform alpha
NCBI Synonym Full Names:nuclear receptor subfamily 3 group C member 1
NCBI Official Symbol:NR3C1
NCBI Official Synonym Symbols:GR; GCR; GRL; GCCR; GCRST
NCBI Protein Information:glucocorticoid receptor
UniProt Protein Name:Glucocorticoid receptor
UniProt Synonym Protein Names:Nuclear receptor subfamily 3 group C member 1
UniProt Gene Name:NR3C1
UniProt Entry Name:GCR_HUMAN
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Additional Information

Product type:
ELISA
Reactivity:
Rat
ELISA Type:
CLIA
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