Human VIL-1 (Villin 1) ELISA Kit (HUES03312)

SKU:
HUES03312
€539

Description

ELISA Kit Technical ManualMSDS

Human VIL1 (Villin 1) ELISA Kit

This ELISA kit uses Sandwich-ELISA as the method. The micro ELISA plate provided in this kit has been pre-coated with an antibody specific to Human VIL1 . Standards or samples are added to the appropriate micro ELISA plate wells and combined with the specific antibody. Then a biotinylated detection antibody specific for Human VIL1 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 VIL1, biotinylated detection antibody and Avidin-HRP conjugate will appear blue in color. The enzyme-substrate reaction is terminated by adding Stop Solution and the color turns yellow. The optical density (OD) is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The OD value is proportional to the concentration of Human VIL1. The concentration of Human VIL1 in samples can be calculated by comparing the OD of the samples to the standard curve.

Assay typeSandwich
Format96T
Assay time4.5h
ReactivityHuman
Detection MethodColormetric
Detection Range0.16-10 ng/mL
Sensitivity0.10 ng/mL
Sample Volume Required Per Well100uL
Sample TypeSerum, plasma and other biological fluids

Specificity

This kit recognizes Human VIL1 in samples. No significant cross-reactivity or interference between Human VIL1 and analogues was observed.

Typical Data

As the OD 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
(ng/mL)
O.D Average Corrected
102.277
2.327
2.3022.229
51.405
1.437
1.4211.348
2.50.868
0.868
0.8680.795
1.250.434
0.442
0.4380.365
0.630.257
0.245
0.2510.178
0.320.174
0.152
0.1630.09
0.160.11
0.13
0.120.047
00.066
0.08
0.073--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human VIL1 were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human VIL1 were tested on 3 different plates, 20 replicates in each plate.

Intra-assay Precision Inter-assay Precision
Sample123123
n202020202020
Mean (ng/mL)0.461.424.830.501.435.01
Standard deviation0.030.060.160.030.080.26
C V (%)6.524.233.316.005.595.19

Recovery

The recovery of Human VIL1 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)91-10597
EDTA plasma (n=5)93-107100
Cell culture media (n=5)87-9994

Linearity

Samples were spiked with high concentrations of Human VIL1 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 (%)99-11591-10591-105
Average (%)1059999
1:4Range (%)87-10082-9487-99
Average (%)948793
1:8Range (%)90-10285-9983-98
Average (%)979190
1:16Range (%)88-10486-9983-94
Average (%)959188

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 ELISA 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 Reagent1 vial, 10 mL4'C(shading light)
Stop Solution1 vial, 10 mL4'C
Plate Sealer5 pieces
Product Description1 copy
Certificate of Analysis1 copy

Human VIL1 (Villin 1) ELISA Kit (HUES03312) 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 90µL of Substrate Reagent to each well. Cover with a new plate seal and incubate forapproximately 15 min at 37°C. Protect the plate from light. Note: the reaction time can beshortened or extended according to the actual color change, but not by more than 30min.
    11. Add 50 µL of Stop Solution to each well. Note: Adding the stop solution should be done inthe same order as the substrate solution.
    12. Determine the optical density (OD value) of each well immediately with a microplate readerset at 450 nm.

Human VIL1 (Villin 1) ELISA Kit (HUES03312) Protein Information

UniProt Protein Function:Villin: Epithelial cell-specific Ca(2+)-regulated actin- modifying protein that modulates the reorganization of microvillar actin filaments. Plays a role in the actin nucleation, actin filament bundle assembly, actin filament capping and severing. Binds phosphatidylinositol 4,5-bisphosphate (PIP2) and lysophosphatidic acid (LPA); binds LPA with higher affinity than PIP2. Binding to LPA increases its phosphorylation by SRC and inhibits all actin-modifying activities. Binding to PIP2 inhibits actin-capping and -severing activities but enhances actin-bundling activity. Regulates the intestinal epithelial cell morphology, cell invasion, cell migration and apoptosis. Protects against apoptosis induced by dextran sodium sulfate (DSS) in the gastrointestinal epithelium. Appears to regulate cell death by maintaining mitochondrial integrity. Enhances hepatocyte growth factor (HGF)-induced epithelial cell motility, chemotaxis and wound repair. Upon S.flexneri cell infection, its actin-severing activity enhances actin-based motility of the bacteria and plays a role during the dissemination. Monomer. Homodimer; homodimerization is necessary for actin-bundling. Associates with F-actin; phosphorylation at tyrosines residues decreases the association with F-actin. Interacts (phosphorylated at C-terminus tyrosine phosphorylation sites) with PLCG1 (via the SH2 domains). Interacts (phosphorylated form) with PLCG1; the interaction is enhanced by hepatocyte growth factor (HGF). Specifically expressed in epithelial cells. Major component of microvilli of intestinal epithelial cells and kidney proximal tubule cells. Expressed in canalicular microvilli of hepatocytes. Belongs to the villin/gelsolin family.
UniProt Protein Details:

Protein type:Actin-binding; Motility/polarity/chemotaxis

Chromosomal Location of Human Ortholog: 2q35

Cellular Component: ruffle; filopodium tip; microvillus; lamellipodium; cytoplasm; actin filament bundle; filopodium

Molecular Function:actin filament binding; identical protein binding; protein binding; phosphatidylinositol-4,5-bisphosphate binding; protein homodimerization activity; caspase inhibitor activity; calcium ion binding

Biological Process: epidermal growth factor receptor signaling pathway; actin filament severing; actin filament depolymerization; actin filament capping; negative regulation of caspase activity; actin nucleation; regulation of cell shape; response to bacterium; epithelial cell differentiation; actin filament polymerization; regulation of actin nucleation; positive regulation of actin filament bundle formation; protein complex assembly; positive regulation of cell migration

NCBI Summary:This gene encodes a member of a family of calcium-regulated actin-binding proteins. This protein represents a dominant part of the brush border cytoskeleton which functions in the capping, severing, and bundling of actin filaments. Two mRNAs of 2.7 kb and 3.5 kb have been observed; they result from utilization of alternate poly-adenylation signals present in the terminal exon. [provided by RefSeq, Jul 2008]
UniProt Code:P09327
NCBI GenInfo Identifier:224471905
NCBI Gene ID:7429
NCBI Accession:P09327.4
UniProt Secondary Accession:P09327,Q53S11, Q96AC8, B2R9A7,
UniProt Related Accession:P09327
Molecular Weight:827
NCBI Full Name:Villin-1
NCBI Synonym Full Names:villin 1
NCBI Official Symbol:VIL1
NCBI Official Synonym Symbols:VIL; D2S1471
NCBI Protein Information:villin-1
UniProt Protein Name:Villin-1
Protein Family:VIN3-like protein
UniProt Gene Name:VIL1
UniProt Entry Name:VILI_HUMAN
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Additional Information

Product type:
ELISA
Reactivity:
Human
ELISA Type:
Sandwich
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