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Canine VE-Cadherin ELISA Kit (CNFI00074)

SKU:
CNFI00074
Product Type:
ELISA Kit
Size:
96 Assays
Reactivity:
Canine
Sensitivity:
<0.281ng/ml
Range:
0.469-30ng/ml
€649
Frequently bought together:

Description

Canine VE-Cadherin ELISA Kit (CNFI00074)

system_update_alt Datasheet system_update_alt MSDS

Key Features

Save Time Pre-coated 96 well plate
Quick Start Kit includes all necessary reagents
Publication Ready Reproducible and reliable results

Overview

Product Name:

Canine VE-Cadherin ELISA Kit

Product Code:

CNFI00074

Size:

96 Assays

Alias:

Vascular Endothelial Cadherin, Cadherin-5, CD144, CDH, 7B4

Uniprot:

A0A8C0TC63

Detection Method:

Sandwich ELISA, Double Antibody

Application:

VE-Cadherin ELISA Kit allows for the in vitro quantitative determination of VE-Cadherin concentrations in serum, plasma, tissue homogenates and other biological fluids.

Sensitivity:

<0.281ng/ml

Range:

0.469-30ng/ml

Storage:

2-8°C for 6 months

Note:

For Research Use Only

Additional Information

Recovery

Matrices listed below were spiked with certain level of VE-Cadherin and the recovery rates were calculated by comparing the measured value to the expected amount of VE-Cadherin in samples.

98

Matrix

Recovery Range (%)

Average (%)

serum(n=5)

88-97

92

EDTA plasma(n=5)

94-103

98

heparin plasma(n=5)

95-103

99

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of VE-Cadherin and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.

Sample

1:2

1:4

1:8

serum(n=5)

86-97%

87-102%

89-102%

EDTA plasma(n=5)

83-92%

91-101%

86-100%

heparin plasma(n=5)

81-97%

80-96%

82-97%

CV(%):

Intra-Assay: CV<8%
Inter-Assay: CV<10%

Kit Components

Component Quantity Storage

ELISA Microplate (Dismountable)

8x12 strips

4°C for 6 months

Lyophilized Standard

2

4°C/ -20°C

Sample/Standard Dlution Buffer

20ml

4°C

Biotin-labeled Antibody (Concentrated)

120ul

4°C (Protection from light)

Antibody Dilution Buffer

10ml

4°C

HRP-Streptavidin Conjugate (SABC)

120ul

4°C (Protect from light)

SABC Dilution Buffer

10ml

4°C

TMB Substrate

10ml

4°C (Protection from light)

Stop Solution

10ml

4°C

Wash Buffer (25X)

30ml

4°C

Plate Sealer

5

-

Other materials required:

  • Microplate reader with 450 nm wavelength filter
  • Multichannel Pipette, Pipette, microcentrifuge tubes and disposable pipette tips
  • Incubator
  • Deionized or distilled water
  • Absorbent paper
  • Buffer resevoir

Protocol

*Note: Protocols are specific to each batch/lot. For the exact instructions please follow the protocol included in your kit.

Before adding to wells, equilibrate the SABC working solution and TMB substrate for at least 30 min at 37°C. When diluting samples and reagents, they must be mixed completely and evenly. It is recommended to plot a standard curve for each test.

Step Procedure

1.

Set standard, test sample and control (zero) wells on the pre-coated plate respectively, and then, record their positions. It is recommended to measure each standard and sample in duplicate. Wash plate 2 times before adding standard, sample and control (zero) wells!

2.

Aliquot 0.1ml standard solutions into the standard wells.

3.

Add 0.1 ml of Sample / Standard dilution buffer into the control (zero) well.

4.

Add 0.1 ml of properly diluted sample ( Human serum, plasma, tissue homogenates and other biological fluids.) into test sample wells.

5.

Seal the plate with a cover and incubate at 37 °C for 90 min.

6.

Remove the cover and discard the plate content, clap the plate on the absorbent filter papers or other absorbent material. Do NOT let the wells completely dry at any time. Wash plate X2.

7.

Add 0.1 ml of Biotin- detection antibody working solution into the above wells (standard, test sample & zero wells). Add the solution at the bottom of each well without touching the side wall.

8.

Seal the plate with a cover and incubate at 37°C for 60 min.

9.

Remove the cover, and wash plate 3 times with Wash buffer. Let wash buffer rest in wells for 1 min between each wash.

10.

Add 0.1 ml of SABC working solution into each well, cover the plate and incubate at 37°C for 30 min.

11.

Remove the cover and wash plate 5 times with Wash buffer, and each time let the wash buffer stay in the wells for 1-2 min.

12.

Add 90 µl of TMB substrate into each well, cover the plate and incubate at 37°C in dark within 10-20 min. (Note: This incubation time is for reference use only, the optimal time should be determined by end user.) And the shades of blue can be seen in the first 3-4 wells (with most concentrated standard solutions), the other wells show no obvious color.

13.

Add 50 µl of Stop solution into each well and mix thoroughly. The color changes into yellow immediately.

14.

Read the O.D. absorbance at 450 nm in a microplate reader immediately after adding the stop solution.

Sample Preparation

When carrying out an ELISA assay it is important to prepare your samples in order to achieve the best possible results. Below we have a list of procedures for the preparation of samples for different sample types.

Sample Type Protocol

Serum

If using serum separator tubes, allow samples to clot for 30 minutes at room temperature. Centrifuge for 10 minutes at 1,000x g. Collect the serum fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles.

If serum separator tubes are not being used, allow samples to clot overnight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Remove serum and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles.

Plasma

Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples at 4°C for 15 mins at 1000 × g within 30 mins of collection. Collect the plasma fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. Note: Over haemolysed samples are not suitable for use with this kit.

Urine & Cerebrospinal Fluid

Collect the urine (mid-stream) in a sterile container, centrifuge for 20 mins at 2000-3000 rpm. Remove supernatant and assay immediately. If any precipitation is detected, repeat the centrifugation step. A similar protocol can be used for cerebrospinal fluid.

Cell culture supernatant

Collect the cell culture media by pipette, followed by centrifugation at 4°C for 20 mins at 1500 rpm. Collect the clear supernatant and assay immediately.

Cell lysates

Solubilize cells in lysis buffer and allow to sit on ice for 30 minutes. Centrifuge tubes at 14,000 x g for 5 minutes to remove insoluble material. Aliquot the supernatant into a new tube and discard the remaining whole cell extract. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.

Tissue homogenates

The preparation of tissue homogenates will vary depending upon tissue type. Rinse tissue with 1X PBS to remove excess blood & homogenize in 20ml of 1X PBS (including protease inhibitors) and store overnight at ≤ -20°C. Two freeze-thaw cycles are required to break the cell membranes. To further disrupt the cell membranes you can sonicate the samples. Centrifuge homogenates for 5 mins at 5000xg. Remove the supernatant and assay immediately or aliquot and store at -20°C or -80°C.

Tissue lysates

Rinse tissue with PBS, cut into 1-2 mm pieces, and homogenize with a tissue homogenizer in PBS. Add an equal volume of RIPA buffer containing protease inhibitors and lyse tissues at room temperature for 30 minutes with gentle agitation. Centrifuge to remove debris. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C

Breast Milk

Collect milk samples and centrifuge at 10,000 x g for 60 min at 4°C. Aliquot the supernatant and assay. For long term use, store samples at -80°C. Minimize freeze/thaw cycles.

Canine VE-Cadherin Background

VE-cadherin, also known as vascular endothelial cadherin or CDH5 (cadherin-5), is a transmembrane protein that plays a critical role in the formation and maintenance of adherens junctions between endothelial cells in blood vessels. It is a member of the cadherin family of cell adhesion molecules.

Function of VE-Cadherin

The primary function of VE-cadherin is to mediate calcium-dependent homophilic cell adhesion between endothelial cells. It acts as a key molecule in the formation of adherens junctions, facilitating the strong and stable attachment between adjacent endothelial cells. This adhesive function of VE-cadherin is critical for the maintenance of endothelial barrier integrity. By forming tight intercellular contacts, VE-cadherin helps to seal the gaps between endothelial cells, preventing the leakage of fluid and molecules across the blood vessel walls. Additionally, VE-cadherin is involved in regulating endothelial cell polarity and migration, which are important processes during angiogenesis, the formation of new blood vessels. Through its adhesive and regulatory functions, VE-cadherin plays a vital role in maintaining the structural integrity and proper functioning of the endothelial lining in blood vessels.

Expression VE-Cadherin

VE-cadherin is predominantly expressed in endothelial cells that line blood vessels. It is specifically localized at the sites of endothelial cell-cell junctions, where it forms adherens junctions. VE-cadherin expression is tightly regulated and is essential for the formation and maintenance of endothelial cell-cell contacts. It is involved in establishing the physical connections between neighboring endothelial cells, contributing to the structural integrity of blood vessels. The expression of VE-cadherin is largely restricted to endothelial cells and is not typically found in other cell types, highlighting its specific role in endothelial cell adhesion.

Regulation of VE-Cadherin

VE-cadherin is regulated by various signaling pathways and can be influenced by factors such as vascular endothelial growth factor (VEGF) and inflammatory mediators. Disruption or dysfunction of VE-cadherin can lead to increased vascular permeability, which is associated with pathological conditions such as inflammation, edema, and tumor metastasis.

Canine VE-Cadherin FAQs

What is the Canine VE-Cadherin ELISA Kit?

The Canine VE-Cadherin ELISA Kit is a diagnostic tool used to measure progesterone levels in biological samples.

What are the advantages of using the Canine VE-Cadherin ELISA Kit?

The Canine VE-Cadherin ELISA Kit offers a sensitive and specific assay platform for accurate quantification of VE-Cadherin in canine samples. It provides reliable and reproducible results, streamlining the experimental workflow with its comprehensive package of components.

Where can I find more information about the Canine VE-Cadherin ELISA Kit?

For more detailed information about the Canine VE-Cadherin ELISA Kit, including technical specifications, performance characteristics, and ordering details, please refer to the product brochure or contact our customer support team. We are here to assist you with any inquiries you may have.

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