Mouse VAV3 / Guanine nucleotide exchange factor VAV3 ELISA Kit

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SKU:
MOFI00207
Product Type:
ELISA Kit
Size:
96 Assays
Uniprot:
Q9R0C8
Sensitivity:
0.094ng/ml
Range:
0.156-10ng/ml
ELISA Type:
Sandwich ELISA, Double Antibody
Synonyms:
Vav3
Reactivity:
Mouse
Frequently bought together:

Description

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Product Name:Mouse VAV3 / Guanine nucleotide exchange factor VAV3 ELISA Kit
Product Code:MOFI00207
Size:96 Assays
Alias:Vav3
Detection Method:Sandwich ELISA
Application:This immunoassay kit allows for the in vitro quantitative determination of Mouse 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 Mouse Vav3 and the recovery rates were calculated by comparing the measured value to the expected amount of Mouse Vav3 in samples.
MatrixRecovery range(%)Average(%)
serum(n=5)87-10094
EDTA plasma(n=5)85-10093
UFH plasma(n=5)85-105100
Linearity:The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Mouse 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)93-105%85-105%95-102%
EDTA plasma(n=5)86-99%87-97%82-97%
UFH plasma(n=5)83-98%85-99%80-97%
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
UniprotQ9R0C8
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:Motility/polarity/chemotaxis; Nuclear receptor co-regulator; Adaptor/scaffold; Actin-binding; GEFs, Rac/Rho; GEFs

Chromosomal Location of Human Ortholog: 1p13.3

Cellular Component: plasma membrane; cytosol

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

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

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:Q9R0C8
NCBI GenInfo Identifier:120432042
NCBI Gene ID:10451
NCBI Accession:NP_001073343.1
UniProt Secondary Accession:Q9R0C8,Q9R0C8,
UniProt Related Accession:Q9UKW4
Molecular Weight:97,776 Da
NCBI Full Name:guanine nucleotide exchange factor VAV3 isoform 2
NCBI Synonym Full Names:vav 3 guanine nucleotide exchange factor
NCBI Official Symbol:VAV3  
NCBI Protein Information:guanine nucleotide exchange factor VAV3; VAV-3; vav 3 oncogene
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.

StepProcedure
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
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.

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