Rat AGER (Advanced Glycosylation End Product Specific Receptor) CLIA Kit (RTES00398)

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SKU:
RTES00398
€649

Description

ELISA Kit Technical ManualMSDS

Rat AGER (Advanced Glycosylation End Product Specific 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 AGER . 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 AGER 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 AGER, 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 AGER. The concentration of Rat AGER 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 range12.50-800 pg/mL
Sensitivity7.50 pg/mL
Sample volume100uL
Sample typeSerum, plasma and other biological fluids
RepeatabilityCV < 15%

Specificity

This kit recognizes Rat AGER in samples. No significant cross-reactivity or interference between Rat AGER 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
80050230
55054
5264252616
40019995
23473
2173421708
2009919
9497
97089682
1004178
4926
45524526
502256
2120
21882162
251140
980
10601034
12.50499
519
509483
026
26
26--

Precision

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

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

  Intra-assay Precision Inter-assay Precision
Sample123123
n202020202020
Mean (pg/mL)41.30122.88267.7539.89130.59287.00
Standard deviation4.8313.8219.494.2310.7723.82
C V (%)11.6911.257.2810.608.258.30

Recovery

The recovery of Rat AGER 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)98-116106
EDTA plasma (n=5)86-10094
Cell culture media (n=5)94-109101

Linearity

Samples were spiked with high concentrations of Rat AGER 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 (%)100-11397-113101-118
Average (%)107103109
1:4Range (%)95-11295-10791-105
Average (%)10210196
1:8Range (%)85-97101-11584-99
Average (%)9210890
1:16Range (%)97-10791-10897-110
Average (%)10299105

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 AGER (Advanced Glycosylation End Product Specific Receptor) CLIA Kit (RTES00398) 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 AGER (Advanced Glycosylation End Product Specific Receptor) CLIA Kit (RTES00398) Protein Information

UniProt Protein Function:RAGE: Mediates interactions of advanced glycosylation end products (AGE). These are nonenzymatically glycosylated proteins which accumulate in vascular tissue in aging and at an accelerated rate in diabetes. Acts as a mediator of both acute and chronic vascular inflammation in conditions such as atherosclerosis and in particular as a complication of diabetes. AGE/RAGE signaling plays an important role in regulating the production/expression of TNF- alpha, oxidative stress, and endothelial dysfunction in type 2 diabetes. Interaction with S100A12 on endothelium, mononuclear phagocytes, and lymphocytes triggers cellular activation, with generation of key proinflammatory mediators. Interaction with S100B after myocardial infarction may play a role in myocyte apoptosis by activating ERK1/2 and p53/TP53 signaling. Receptor for amyloid beta peptide. Contributes to the translocation of amyloid-beta peptide (ABPP) across the cell membrane from the extracellular to the intracellular space in cortical neurons. ABPP-initiated RAGE signaling, especially stimulation of p38 mitogen-activated protein kinase (MAPK), has the capacity to drive a transport system delivering ABPP as a complex with RAGE to the intraneuronal space. Interacts with S100B, S100A1 and APP. Interacts with S100A12. Endothelial cells. 4 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:

Protein type:Cell cycle regulation; Receptor, misc.; Motility/polarity/chemotaxis; Membrane protein, integral

Cellular Component: extracellular space; cell surface; cell soma; axon; cytoplasm; integral to membrane; basal plasma membrane; plasma membrane; cytosol

Molecular Function:identical protein binding; protein binding; advanced glycation end-product receptor activity

Biological Process: positive regulation of JNK activity; glycoprotein metabolic process; positive regulation of apoptosis; positive regulation of smooth muscle cell proliferation; negative regulation of collagen biosynthetic process; negative regulation of cell adhesion; positive regulation of smooth muscle cell migration; induction of positive chemotaxis; response to vitamin A; activation of NF-kappaB transcription factor; calcium ion homeostasis; positive regulation of fibroblast proliferation; negative regulation of protein amino acid phosphorylation; regulation of DNA binding; positive regulation of neuron apoptosis; response to methylglyoxal; inflammatory response; positive regulation of autophagy; response to fructose stimulus; neurite development; JAK-STAT cascade; response to genistein; response to hyperoxia; regulation of inflammatory response; negative regulation of endothelial cell proliferation; response to hypoxia; negative regulation of osteoblast proliferation; brain development; positive regulation of protein amino acid phosphorylation; response to activity; positive regulation of phagocytosis, engulfment; lung development; positive regulation of inflammatory response; positive regulation of cell migration

NCBI Summary:cell surface receptor for various molecules, including the amyloidogenic form of serum amyloid A [RGD, Feb 2006]
UniProt Code:Q63495
NCBI GenInfo Identifier:2497319
NCBI Gene ID:81722
NCBI Accession:Q63495.1
UniProt Related Accession:Q63495
Molecular Weight:42,664 Da
NCBI Full Name:Advanced glycosylation end product-specific receptor
NCBI Synonym Full Names:advanced glycosylation end product-specific receptor
NCBI Official Symbol:Ager  
NCBI Official Synonym Symbols:RAGE  
NCBI Protein Information:advanced glycosylation end product-specific receptor; receptor for advanced glycosylation end products; advanced glycosylation end product-specific receptor variant 2; advanced glycosylation end product-specific receptor variant 3; advanced glycosylation end product-specific receptor variant 4; advanced glycosylation end product-specific receptor variant 5
UniProt Protein Name:Advanced glycosylation end product-specific receptor
UniProt Synonym Protein Names:Receptor for advanced glycosylation end products
Protein Family:Advanced glycosylation end product-specific receptor
UniProt Gene Name:Ager  
UniProt Entry Name:RAGE_RAT
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Additional Information

Product type:
ELISA
Reactivity:
Rat
ELISA Type:
CLIA
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