Human BCAR1 (Breast Cancer Anti Estrogen Resistance 1) CLIA Kit (HUES01230)

SKU:
HUES01230
Product type:
ELISA
Reactivity:
Human
ELISA Type:
CLIA
€599

Description

ELISA Kit Technical ManualMSDS

Human BCAR1 (Breast Cancer Anti Estrogen Resistance 1) 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 Human BCAR1 . 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 Human BCAR1 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 Human BCAR1, 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 Human BCAR1. The concentration of Human BCAR1 in the samples can be calculated by comparing the RLU of the samples to the standard curve.

Assay typeSandwich
Format96T
Assay time4.5h
ReactivityHuman
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 Human BCAR1 in samples. No significant cross-reactivity or interference between Human BCAR1 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
200054528
56914
5572155693
100023898
24050
2397423946
50011227
10837
1103211004
2505208
5380
52945266
1252664
2552
26082580
62.51403
1219
13111283
31.25661
687
674646
027
29
28--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human BCAR1 were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human BCAR1 were tested on 3 different plates, 20 replicates in each plate.

  Intra-assay Precision Inter-assay Precision
Sample123123
n202020202020
Mean (pg/mL)95.90284.94882.6490.87270.63890.03
Standard deviation8.2123.1994.8010.8728.0497.90
C V (%)8.568.1410.7411.9610.3611.00

Recovery

The recovery of Human BCAR1 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)93-107101
EDTA plasma (n=5)99-113106
Cell culture media (n=5)95-106101

Linearity

Samples were spiked with high concentrations of Human BCAR1 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 (%)94-11097-11185-100
Average (%)10210392
1:4Range (%)93-106102-116101-113
Average (%)98110107
1:8Range (%)99-111103-11994-106
Average (%)105109100
1:16Range (%)95-11097-11289-100
Average (%)10110494

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

Human BCAR1 (Breast Cancer Anti Estrogen Resistance 1) CLIA Kit (HUES01230) 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 100 µL of standard solutions into the standard wells.
    3. Add 100 µL of Sample / Standard dilution buffer into the control (zero) well.
    4. Add 100 µL 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 100 µL 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 350 µL 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 100 µL 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 100 µL 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.

Human BCAR1 (Breast Cancer Anti Estrogen Resistance 1) CLIA Kit (HUES01230) Protein Information

UniProt Protein Function:P130Cas: a docking protein containing multiple protein-protein interaction domains. Plays a central coordinating role for tyrosine-kinase-based signaling related to cell adhesion. Implicated in induction of cell migration. The amino-terminal SH3 domain regulates its interaction with focal adhesion kinase (FAK) and the FAK-related kinase PYK2 and also with tyrosine phosphatases PTP-1B and PTP-PEST. Overexpression confers antiestrogen resistance on breast cancer cells.
UniProt Protein Details:

Protein type:Motility/polarity/chemotaxis; Adaptor/scaffold

Chromosomal Location of Human Ortholog: 16q23.1

Cellular Component: ruffle; focal adhesion; lamellipodium; cytoplasm; nucleolus; plasma membrane; cytosol; actin cytoskeleton

Molecular Function:signal transducer activity; protein binding; SH3 domain binding; protein kinase binding

Biological Process: epidermal growth factor receptor signaling pathway; integrin-mediated signaling pathway; antigen receptor-mediated signaling pathway; platelet activation; cell migration; nerve growth factor receptor signaling pathway; actin filament organization; hepatocyte growth factor receptor signaling pathway; platelet-derived growth factor receptor signaling pathway; T cell receptor signaling pathway; regulation of apoptosis; G-protein coupled receptor protein signaling pathway; cell proliferation; B cell receptor signaling pathway; cell division; insulin receptor signaling pathway; regulation of cell growth; cell adhesion; vascular endothelial growth factor receptor signaling pathway; blood coagulation; positive regulation of cell migration

NCBI Summary:BCAR1, or CAS, is an Src (MIM 190090) family kinase substrate involved in various cellular events, including migration, survival, transformation, and invasion (Sawada et al., 2006 [PubMed 17129785]).[supplied by OMIM, May 2009]
UniProt Code:P56945
NCBI GenInfo Identifier:288558806
NCBI Gene ID:9564
NCBI Accession:P56945.2
UniProt Secondary Accession:P56945,B3KWD7, B4DEV4, B4DGB5, B4DIW5, B7Z7X7, E9PCL5 E9PCV2, F5GXA2, F5GXV6, F5H7Z0, F8WA69,
UniProt Related Accession:P56945
Molecular Weight:870
NCBI Full Name:Breast cancer anti-estrogen resistance protein 1
NCBI Synonym Full Names:breast cancer anti-estrogen resistance 1
NCBI Official Symbol:BCAR1
NCBI Official Synonym Symbols:CAS; CAS1; CASS1; CRKAS; P130Cas
NCBI Protein Information:breast cancer anti-estrogen resistance protein 1; Crk-associated substrate p130Cas; Cas scaffolding protein family member 1
UniProt Protein Name:Breast cancer anti-estrogen resistance protein 1
UniProt Synonym Protein Names:CRK-associated substrate; Cas scaffolding protein family member 1; p130cas
Protein Family:Breast cancer anti-estrogen resistance protein
UniProt Gene Name:BCAR1
UniProt Entry Name:BCAR1_HUMAN
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