Description
Human VCL (Vinculin) ELISA Kit
The Human VCL (Vinculin) ELISA Kit is specifically designed for the precise quantification of Vinculin levels in human serum, plasma, and cell culture supernatants. This kit offers exceptional sensitivity and specificity, guaranteeing consistent and accurate results, making it perfect for various research purposes.Vinculin is a key protein involved in cell adhesion and cytoskeleton organization, crucial for maintaining cell structure and function.
Dysregulation of Vinculin has been linked to various diseases, including cancer and cardiovascular disorders, highlighting its importance as a biomarker for disease progression and therapeutic development. By using the Human VCL (Vinculin) ELISA Kit, researchers can effectively investigate the role of Vinculin in different physiological and pathological processes, paving the way for innovative treatment strategies and diagnostic advancements.
| Assay type: | Sandwich |
| Format: | 96T |
| Assay time: | 4.5h |
| Reactivity: | Human |
| Detection Method: | Colormetric |
| Detection Range: | 78.13-5000 pg/mL |
| Sensitivity: | 46.88 pg/mL |
| Sample Volume Required Per Well: | 100µL |
| Sample Type: | Serum, plasma and other biological fluids |
| Specificity: | This kit recognizes Human VCL in samples. No significant cross-reactivity or interference between Human VCL and analogues was observed. |
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 VCL. 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 VCL 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 VCL, 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 VCL. The concentration of Human VCL in samples can be calculated by comparing the OD of the samples to the standard curve.
| UniProt Protein Function: | Vinculin: Actin filament (F-actin)-binding protein involved in cell-matrix adhesion and cell-cell adhesion. Regulates cell- surface E-cadherin expression and potentiates mechanosensing by the E-cadherin complex. May also play important roles in cell morphology and locomotion. Exhibits self-association properties. Interacts with NRAP and SORBS1. Interacts with TLN1. Interacts with SYNM. Interacts with CTNNB1 and this interaction is necessary for its localization to the cell-cell junctions and for its function in regulating cell surface expression of E-cadherin. Metavinculin is muscle-specific. Belongs to the vinculin/alpha-catenin family. 3 isoforms of the human protein are produced by alternative splicing. |
| UniProt Protein Details: | Protein type:Cell adhesion; Cytoskeletal; Motility/polarity/chemotaxis Chromosomal Location of Human Ortholog: 10q22. 2 Cellular Component: cytoskeleton; adherens junction; protein complex; cell-cell adherens junction; focal adhesion; costamere; plasma membrane; extracellular region; fascia adherens; intercellular junction; cytosol; vesicle; cell-matrix junction; actin cytoskeleton Molecular Function:protein binding; cadherin binding; beta-catenin binding; structural molecule activity; actin binding; alpha-catenin binding Biological Process: lamellipodium biogenesis; platelet activation; platelet degranulation; apical junction assembly; muscle contraction; axon extension; cell-matrix adhesion; morphogenesis of an epithelium; cell adhesion; cell motility; blood coagulation; negative regulation of cell migration Disease: Cardiomyopathy, Dilated, 1w |
| NCBI Summary: | Vinculin is a cytoskeletal protein associated with cell-cell and cell-matrix junctions, where it is thought to function as one of several interacting proteins involved in anchoring F-actin to the membrane. Defects in VCL are the cause of cardiomyopathy dilated type 1W. Dilated cardiomyopathy is a disorder characterized by ventricular dilation and impaired systolic function, resulting in congestive heart failure and arrhythmia. Multiple alternatively spliced transcript variants have been found for this gene, but the biological validity of some variants has not been determined. [provided by RefSeq, Jul 2008] |
| UniProt Code: | P18206 |
| NCBI GenInfo Identifier: | 21903479 |
| NCBI Gene ID: | 7414 |
| NCBI Accession: | P18206. 4 |
| UniProt Secondary Accession: | P18206,Q16450, Q5SWX2, Q7Z3B8, Q8IXU7, |
| UniProt Related Accession: | P18206 |
| Molecular Weight: | 24,904 Da |
| NCBI Full Name: | Vinculin |
| NCBI Synonym Full Names: | vinculin |
| NCBI Official Symbol: | VCL |
| NCBI Official Synonym Symbols: | MV; MVCL; CMD1W; CMH15; HEL114 |
| NCBI Protein Information: | vinculin; metavinculin; epididymis luminal protein 114 |
| UniProt Protein Name: | Vinculin |
| UniProt Synonym Protein Names: | Metavinculin; MV |
| Protein Family: | Vinculin |
| UniProt Gene Name: | VCL |
| UniProt Entry Name: | VINC_HUMAN |
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 (pg/mL) | O.D | Average | Corrected |
| 5000 | 2.463 2.473 | 2.468 | 2.399 |
| 2500 | 1.792 1.816 | 1.804 | 1.735 |
| 1250 | 1.073 1.065 | 1.069 | 1 |
| 625 | 0.508 0.546 | 0.527 | 0.458 |
| 312.5 | 0.267 0.241 | 0.254 | 0.185 |
| 156.25 | 0.181 0.179 | 0.18 | 0.111 |
| 78.13 | 0.122 0.128 | 0.125 | 0.056 |
| 0 | 0.065 0.073 | 0.069 | -- |
Precision
Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human VCL were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human VCL were tested on 3 different plates, 20 replicates in each plate.
| Intra-assay Precision | Inter-assay Precision | |||||
| Sample | 1 | 2 | 3 | 1 | 2 | 3 |
| n | 20 | 20 | 20 | 20 | 20 | 20 |
| Mean (pg/mL) | 230.45 | 576.53 | 2280.42 | 245.01 | 555.49 | 2218.29 |
| Standard deviation | 13.25 | 32.75 | 97.60 | 15.39 | 23.00 | 70.54 |
| C V (%) | 5.75 | 5.68 | 4.28 | 6.28 | 4.14 | 3.18 |
Recovery
The recovery of Human VCL 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) | 90-104 | 97 |
| EDTA plasma (n=5) | 85-99 | 91 |
| Cell culture media (n=5) | 90-105 | 96 |
Linearity
Samples were spiked with high concentrations of Human VCL 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) | ||
| 1:2 | Range (%) | 97-110 | 93-108 | 90-105 |
| Average (%) | 104 | 99 | 96 | |
| 1:4 | Range (%) | 91-104 | 87-98 | 81-93 |
| Average (%) | 97 | 92 | 88 | |
| 1:8 | Range (%) | 89-102 | 86-99 | 87-101 |
| Average (%) | 95 | 92 | 94 | |
| 1:16 | Range (%) | 90-104 | 80-93 | 83-93 |
| Average (%) | 95 | 86 | 88 |
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 ×12 strips | -20°C, 6 months |
| Reference Standard | 2 vials | |
| Concentrated Biotinylated Detection Ab (100×) | 1 vial, 120 µL | |
| Concentrated HRP Conjugate (100×) | 1 vial, 120 µL | -20°C(shading light), 6 months |
| Reference Standard & Sample Diluent | 1 vial, 20 mL | 4°C, 6 months |
| Biotinylated Detection Ab Diluent | 1 vial, 14 mL | |
| HRP Conjugate Diluent | 1 vial, 14 mL | |
| Concentrated Wash Buffer (25×) | 1 vial, 30 mL | |
| Substrate Reagent | 1 vial, 10 mL | 4°C(shading light) |
| Stop Solution | 1 vial, 10 mL | 4°C |
| Plate Sealer | 5 pieces | |
| Product Description | 1 copy | |
| Certificate of Analysis | 1 copy |
- 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.
- Aliquot 100µl of standard solutions into the standard wells.
- Add 100µl of Sample / Standard dilution buffer into the control (zero) well.
- Add 100µl of properly diluted sample (serum, plasma, tissue homogenates and otherbiological fluids) into test sample wells.
- Cover the plate with the sealer provided in the kit and incubate for 90 min at 37°C.
- 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.
- 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.
- Add 100µL of HRP Conjugate working solution to each well. Cover with a plate seal andincubate for 30 min at 37°C.
- Aspirate or decant the solution from each well. Repeat the wash process for five times asconducted in step 7.
- 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.
- Add 50 µL of Stop Solution to each well. Note: Adding the stop solution should be done inthe same order as the substrate solution.
- Determine the optical density (OD value) of each well immediately with a microplate readerset at 450 nm.
