Human VAV3 / Guanine nucleotide exchange factor VAV3 ELISA Kit

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SKU:
HUFI00564
Product Type:
ELISA Kit
Size:
96 Assays
Uniprot:
Q9UKW4
Sensitivity:
0.094ng/ml
Range:
0.156-10ng/ml
ELISA Type:
Sandwich ELISA, Double Antibody
Synonyms:
VAV3, Guanine nucleotide exchange factor VAV3, VAV-3
Reactivity:
Human
Frequently bought together:

Description

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Product Name:Human VAV3 / Guanine nucleotide exchange factor VAV3 ELISA Kit
Product Code:HUFI00564
Size:96 Assays
Alias:VAV3, Guanine nucleotide exchange factor VAV3, VAV-3
Detection method:Sandwich ELISA, Double Antibody
Application:This immunoassay kit allows for the in vitro quantitative determination of Human VAV3 concentrations in serum plasma and other biological fluids.
Sensitivity:0.094ng/ml
Range:0.156-10ng/ml
Storage:4°C for 6 months
Note:For Research Use Only
Recovery:Matrices listed below were spiked with certain level of Human VAV3 and the recovery rates were calculated by comparing the measured value to the expected amount of Human VAV3 in samples.
MatrixRecovery range(%)Average(%)
serum(n=5)90-10296
EDTA plasma(n=5)95-10197
UFH plasma(n=5)87-10294
Linearity:The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human VAV3 and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.
Sample1:21:41:8
serum(n=5)87-103%92-103%86-105%
EDTA plasma(n=5)82-100%82-96%85-96%
UFH plasma(n=5)89-97%84-98%85-100%
CV(%):Intra-Assay: CV<8%
Inter-Assay: CV<10%
ComponentQuantityStorage
ELISA Microplate (Dismountable)8×12 strips4°C for 6 months
Lyophilized Standard24°C/-20°C
Sample/Standard Dilution Buffer20ml4°C
Biotin-labeled Antibody(Concentrated)120ul4°C (Protect from light)
Antibody Dilution Buffer10ml4°C
HRP-Streptavidin Conjugate(SABC)120ul4°C (Protect from light)
SABC Dilution Buffer10ml4°C
TMB Substrate10ml4°C (Protect from light)
Stop Solution10ml4°C
Wash Buffer(25X)30ml4°C
Plate Sealer5-

Other materials and equipment 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
UniprotQ9UKW4
UniProt Protein Function:VAV3: a guanine nucleotide exchange factor (GEF) that activates RhoA, RhoG and, to a lesser extent, Rac1. Binds physically to the nucleotide-free states of those GTPases. Plays an important role in angiogenesis. Its recruitment by activated EPHA2 is critical for EFNA1-induced RAC1 GTPase activation and vascular endothelial cell migration and assembly. May be important for integrin-mediated signaling, at least in some cell types. In osteoclasts, along with SYK tyrosine kinase, required for signaling through integrin alpha-v/beta-1 (ITAGV-ITGB1), a crucial event for osteoclast proper cytoskeleton organization and function. This signaling pathway involves RAC1, but not RHO, activation. Necessary for proper wound healing. In the course of wound healing, required for the phagocytotic cup formation preceding macrophage phagocytosis of apoptotic neutrophils. Responsible for integrin beta-2 (ITGB2)-mediated macrophage adhesion and, to a lesser extent, contributes to beta-3 (ITGB3)-mediated adhesion. Does not affect integrin beta-1 (ITGB1)-mediated adhesion. Interacts with the PH domain of APS. Interacts (via SH2 domains) with the phosphorylated form of EPHA2. Interacts with ROS1; constitutive interaction that mediates VAV3 phosphorylation. Down-regulated by EGF and TGF-beta. Four isoforms of the human protein are produced by alternative promoter. Isoform 1 and isoform 3 are widely expressed; both are expressed at very low levels in skeletal muscle. In keratinocytes, isoform 1 is less abundant than isoform 3. Isoform 3 is detected at very low levels, if any, in adrenal gland, bone marrow, spleen, fetal brain and spinal chord; in these tissues, isoform 1 is readily detectable.
UniProt Protein Details:Protein type:Nuclear receptor co-regulator; Actin-binding; Motility/polarity/chemotaxis; Adaptor/scaffold; GEFs, Rac/Rho; GEFs

Chromosomal Location of Human Ortholog: 1p13.3

Cellular Component: cytosol; plasma membrane

Molecular Function:epidermal growth factor receptor binding; GTPase activator activity; guanyl-nucleotide exchange factor activity; metal ion binding; protein binding; Rac guanyl-nucleotide exchange factor activity; SH3/SH2 adaptor activity

Biological Process: angiogenesis; axon guidance; B cell receptor signaling pathway; blood coagulation; ephrin receptor signaling pathway; innate immune response; integrin-mediated signaling pathway; lamellipodium biogenesis; nerve growth factor receptor signaling pathway; neutrophil chemotaxis; platelet activation; positive regulation of apoptosis; positive regulation of B cell proliferation; positive regulation of cell adhesion; positive regulation of GTPase activity; positive regulation of phosphoinositide 3-kinase activity; positive regulation of signal transduction; regulation of cell size; regulation of GTPase activity; regulation of Rho protein signal transduction; regulation of small GTPase mediated signal transduction; response to DNA damage stimulus; response to drug; small GTPase mediated signal transduction; vascular endothelial growth factor receptor signaling pathway; vesicle fusion

NCBI Summary:This gene is a member of the VAV gene family. The VAV proteins are guanine nucleotide exchange factors (GEFs) for Rho family GTPases that activate pathways leading to actin cytoskeletal rearrangements and transcriptional alterations. This gene product acts as a GEF preferentially for RhoG, RhoA, and to a lesser extent, RAC1, and it associates maximally with the nucleotide-free states of these GTPases. Alternatively spliced transcript variants encoding different isoforms have been described for this gene. [provided by RefSeq, Jul 2008]
UniProt Code:Q9UKW4
NCBI GenInfo Identifier:12643372
NCBI Gene ID:10451
NCBI Accession:Q9UKW4.1
UniProt Secondary Accession:Q9UKW4,O60498, O95230, Q9Y5X8, B1AMM0, B1APV5, B4E232 B7ZLR1, E9PQ97,
UniProt Related Accession:Q9UKW4
Molecular Weight:100,828 Da
NCBI Full Name:Guanine nucleotide exchange factor VAV3
NCBI Synonym Full Names:vav guanine nucleotide exchange factor 3
NCBI Official Symbol:VAV3
NCBI Protein Information:guanine nucleotide exchange factor VAV3
UniProt Protein Name:Guanine nucleotide exchange factor VAV3
Protein Family:Guanine nucleotide exchange factor
UniProt Gene Name:VAV3
UniProt Entry Name:VAV3_HUMAN

*Note: Protocols are specific to each batch/lot. For the correct 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.

StepProtocol
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.

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 TypeProtocol
SerumIf 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.

PlasmaCollect 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 FluidCollect 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 supernatantCollect 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 lysatesSolubilize 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 homogenatesThe 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 lysatesRinse 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 MilkCollect 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.

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