Rat HSP-90 (Heat Shock Protein 90) CLIA Kit (RTES00280)

SKU:
RTES00280
Product type:
ELISA
Reactivity:
Rat
ELISA Type:
CLIA
€649

Description

ELISA Kit Technical ManualMSDS

Rat HSP-90 (Heat Shock Protein 90) 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 HSP-90 . 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 HSP-90 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 HSP-90, 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 HSP-90. The concentration of Rat HSP-90 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 HSP-90 in samples. No significant cross-reactivity or interference between Rat HSP-90 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
200052258
59184
5572155693
100023375
24573
2397423946
50011840
10224
1103211004
2505198
5390
52945266
1252775
2441
26082580
62.51320
1302
13111283
31.25638
710
674646
027
29
28--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Rat HSP-90 were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Rat HSP-90 were tested on 3 different plates, 20 replicates in each plate.

  Intra-assay Precision Inter-assay Precision
Sample123123
n202020202020
Mean (pg/mL)97.84171.97837.39105.44163.68900.90
Standard deviation8.9017.4053.6812.4716.9793.51
C V (%)9.1010.126.4111.8310.3710.38

Recovery

The recovery of Rat HSP-90 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)85-9790
EDTA plasma (n=5)96-110102
Cell culture media (n=5)98-110104

Linearity

Samples were spiked with high concentrations of Rat HSP-90 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 (%)86-10295-10891-108
Average (%)9310198
1:4Range (%)94-11090-10593-106
Average (%)1029898
1:8Range (%)94-11196-11299-110
Average (%)101103104
1:16Range (%)87-100101-11588-100
Average (%)9410794

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 HSP-90 (Heat Shock Protein 90) CLIA Kit (RTES00280) 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 HSP-90 (Heat Shock Protein 90) CLIA Kit (RTES00280) Protein Information

UniProt Protein Function:HSP90A: a molecular chaperone of the heat shock protein 90 family. Has ATPase activity. Known to interact with a wide variety of proteins including steroid hormone receptors, neuropeptide Y, FKBP51/54, and FKBP52. G protein-coupled receptor kinases are stabilized by interacting with HSP 90. Hsp70 and Hsp90 promote tau solubility and tau binding to microtubules, reducing insoluble tau phosphorylation of tau.
UniProt Protein Details:

Protein type:Heat shock protein; Chaperone

Chromosomal Location of Human Ortholog: 14q32.33

Cellular Component: nucleoplasm; membrane; mitochondrion; cytoplasm; extracellular region; plasma membrane; melanosome; nucleus; cytosol

Molecular Function:identical protein binding; protein binding; protein homodimerization activity; TPR domain binding; ATPase activity; nitric-oxide synthase regulator activity; unfolded protein binding; nucleotide binding; ATP binding

Biological Process: axon guidance; receptor-mediated endocytosis; positive regulation of nitric oxide biosynthetic process; organelle organization and biogenesis; signal transduction; nitric oxide metabolic process; protein import into mitochondrial outer membrane; response to unfolded protein; mitochondrial transport; innate immune response; protein refolding; mitotic cell cycle; regulation of nitric-oxide synthase activity; vascular endothelial growth factor receptor signaling pathway; G2/M transition of mitotic cell cycle; chaperone-mediated protein complex assembly

NCBI Summary:The protein encoded by this gene is an inducible molecular chaperone that functions as a homodimer. The encoded protein aids in the proper folding of specific target proteins by use of an ATPase activity that is modulated by co-chaperones. Two transcript variants encoding different isoforms have been found for this gene. [provided by RefSeq, Jan 2012]
UniProt Code:P34058
NCBI GenInfo Identifier:153792590
NCBI Gene ID:3320
NCBI Accession:NP_001017963.2
UniProt Secondary Accession:P34058,P34058, P11499, Q4R4T5, P08238, O57521, Q76LV1 P82995, P30946, O02705, P07901, Q4R4P1, P46633, P11501 Q76LV2
UniProt Related Accession:P07900
Molecular Weight:84660
NCBI Full Name:heat shock protein HSP 90-alpha isoform 1
NCBI Synonym Full Names:heat shock protein 90 alpha family class A member 1
NCBI Official Symbol:HSP90AA1
NCBI Official Synonym Symbols:EL52; HSPN; LAP2; HSP86; HSPC1; HSPCA; Hsp89; Hsp90; LAP-2; HSP89A; HSP90A; HSP90N; Hsp103; HSPCAL1; HSPCAL4; HEL-S-65p
NCBI Protein Information:heat shock protein HSP 90-alpha
UniProt Protein Name:Heat shock protein HSP 90-alpha
UniProt Synonym Protein Names:Heat shock 86 kDa; HSP 86; HSP86; Renal carcinoma antigen NY-REN-38
Protein Family:Heat shock protein
UniProt Gene Name:HSP90AA1
UniProt Entry Name:HS90A_HUMAN
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