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VAV1 (Phospho-Tyr174) Colorimetric Cell-Based ELISA Kit

SKU:
CBCAB00404
Product Type:
ELISA Kit
ELISA Type:
Cell Based Phospho Specific
Reactivity:
Human
Mouse
Rat
Detection Method:
Colorimetric
€599
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Description

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VAV1 (Phospho-Tyr174)Colorimetric Cell-Based ELISA Kit

The VAV1 (Phospho Tyr174) Colorimetric Cell-Based ELISA Kit is an innovative assay designed for the accurate measurement of VAV1 phosphorylation at tyrosine 174 in cell lysates. This kit offers high sensitivity and specificity, allowing researchers to obtain reliable and reproducible results for studying the activation of VAV1 signaling pathways.VAV1 is a key signaling protein involved in T cell activation, cytokine signaling, and cell migration. Phosphorylation of VAV1 at tyrosine 174 plays a crucial role in regulating its activity and function in various cellular processes.

Dysregulation of VAV1 phosphorylation has been implicated in immune disorders, cancer, and inflammatory diseases, highlighting the importance of studying this protein for potential therapeutic interventions.With its user-friendly protocol and efficient colorimetric detection system, the VAV1 (Phospho Tyr174) ELISA Kit is ideal for researchers investigating the role of VAV1 signaling in health and disease. Unlock the potential of VAV1 phosphorylation analysis with this cutting-edge cell-based ELISA kit from Assay Genie.

Product Name:VAV1 (Phospho-Tyr174) Colorimetric Cell-Based ELISA
Product Code:CBCAB00404
ELISA Type:Cell-Based
Target:VAV1 (Phospho-Tyr174)
Reactivity:Human, Mouse, Rat
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nm
Format:2 x 96-Well Microplates

The VAV1 (Phospho-Tyr174) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect VAV1 protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated VAV1 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on VAV1 phosphorylation.

Qualitative determination of VAV1 (Phospho-Tyr174) concentration is achieved by an indirect ELISA format. In essence, VAV1 (Phospho-Tyr174) is captured by VAV1 (Phospho-Tyr174)-specific primary antibodies while the HRP-conjugated secondary antibodies bind the Fc region of the primary antibody. Through this binding, the HRP enzyme conjugated to the secondary antibody can catalyze a colorimetric reaction upon substrate addition. Due to the qualitative nature of the Cell-Based ELISA, multiple normalization methods are needed:

1. A monoclonal antibody specific for human GAPDH is included to serve as an internal positive control in normalizing the target absorbance values.
2. Following the colorimetric measurement of HRP activity via substrate addition, the Crystal Violet whole-cell staining method may be used to determine cell density. After staining, the results can be analysed by normalizing the absorbance values to cell amounts, by which the plating difference can be adjusted.
Database Information:Gene ID: 7409, UniProt ID: P15498, OMIM: 164875, Unigene: Hs.116237
Gene Symbol:VAV
Sub Type:Phospho
UniProt Protein Function:VAV1: a proto-oncogenic guanine nucleotide exchange factors (GEF) of the Dbl family. A GEF for the Rho family of GTP binding proteins. Expressed exclusively in hematopoietic cells. Important in hematopoiesis, playing a role in T-cell and B-cell development and activation.
UniProt Protein Details:

Protein type:GEFs, Rac/Rho; GEFs; Oncoprotein; Motility/polarity/chemotaxis

Chromosomal Location of Human Ortholog: 19p13.2

Cellular Component: plasma membrane; intercellular junction; cytosol

Molecular Function:protein binding; metal ion binding; Rac guanyl-nucleotide exchange factor activity; guanyl-nucleotide exchange factor activity; transcription factor activity

Biological Process: epidermal growth factor receptor signaling pathway; integrin-mediated signaling pathway; neutrophil chemotaxis; platelet activation; phosphoinositide-mediated signaling; fibroblast growth factor receptor signaling pathway; T cell activation; nerve growth factor receptor signaling pathway; positive regulation of apoptosis; G-protein coupled receptor protein signaling pathway; regulation of small GTPase mediated signal transduction; regulation of transcription, DNA-dependent; small GTPase mediated signal transduction; T cell costimulation; innate immune response; blood coagulation; vascular endothelial growth factor receptor signaling pathway

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. The encoded protein is important in hematopoiesis, playing a role in T-cell and B-cell development and activation. The encoded protein has been identified as the specific binding partner of Nef proteins from HIV-1. Coexpression and binding of these partners initiates profound morphological changes, cytoskeletal rearrangements and the JNK/SAPK signaling cascade, leading to increased levels of viral transcription and replication. Alternatively spliced transcript variants encoding multiple isoforms have been observed for this gene. [provided by RefSeq, Apr 2012]
UniProt Code:P15498
NCBI GenInfo Identifier:13124807
NCBI Gene ID:7409
NCBI Accession:P15498.4
UniProt Secondary Accession:P15498,Q15860, B4DVK9, M0QXX6,
UniProt Related Accession:P15498
Molecular Weight:845
NCBI Full Name:Proto-oncogene vav
NCBI Synonym Full Names:vav 1 guanine nucleotide exchange factor
NCBI Official Symbol:VAV1  
NCBI Official Synonym Symbols:VAV  
NCBI Protein Information:proto-oncogene vav; vav 1 oncogene
UniProt Protein Name:Proto-oncogene vav
Protein Family:Proto-oncogene
UniProt Gene Name:VAV1  
UniProt Entry Name:VAV_HUMAN
ComponentQuantity
96-Well Cell Culture Clear-Bottom Microplate2 plates
10X TBS24 mL
Quenching Buffer24 mL
Blocking Buffer50 mL
15X Wash Buffer50 mL
Primary Antibody Diluent12 mL
100x Anti-Phospho Target Antibody 60 µL
100x Anti-Target Antibody60 µL
Anti-GAPDH Antibody60 µL
HRP-Conjugated Anti-Rabbit IgG Antibody12 mL
HRP-Conjugated Anti-Mouse IgG Antibody12 mL
SDS Solution12 mL
Stop Solution24 mL
Ready-to-Use Substrate12 mL
Crystal Violet Solution12 mL
Adhesive Plate Seals2 seals

The following materials and/or equipment are NOT provided in this kit but are necessary to successfully conduct the experiment:

  • Microplate reader able to measure absorbance at 450 nm and/or 595 nm for Crystal Violet Cell Staining (Optional)
  • Micropipettes with capability of measuring volumes ranging from 1 µL to 1 ml
  • 37% formaldehyde (Sigma Cat# F-8775) or formaldehyde from other sources
  • Squirt bottle, manifold dispenser, multichannel pipette reservoir or automated microplate washer
  • Graph paper or computer software capable of generating or displaying logarithmic functions
  • Absorbent papers or vacuum aspirator
  • Test tubes or microfuge tubes capable of storing ≥1 ml
  • Poly-L-Lysine (Sigma Cat# P4832 for suspension cells)
  • Orbital shaker (optional)
  • Deionized or sterile water

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

StepProcedure
1.Seed 200 µL of 20,000 adherent cells in culture medium in each well of a 96-well plate. The plates included in the kit are sterile and treated for cell culture. For suspension cells and loosely attached cells, coat the plates with 100 µL of 10 µg/ml Poly-L-Lysine (not included) to each well of a 96-well plate for 30 minutes at 37°C prior to adding cells.
2.Incubate the cells for overnight at 37°C, 5% CO2.
3.Treat the cells as desired.
4.Remove the cell culture medium and rinse with 200 µL of 1x TBS, twice.
5.Fix the cells by incubating with 100 µL of Fixing Solution for 20 minutes at room temperature. The 4% formaldehyde is used for adherent cells and 8% formaldehyde is used for suspension cells and loosely attached cells.
6.Remove the Fixing Solution and wash the plate 3 times with 200 µL 1x Wash Buffer for five minutes each time with gentle shaking on the orbital shaker. The plate can be stored at 4°C for a week.
7.Add 100 µL of Quenching Buffer and incubate for 20 minutes at room temperature.
8.Wash the plate 3 times with 1x Wash Buffer for 5 minutes each time.
9.Add 200 µL of Blocking Buffer and incubate for 1 hour at room temperature.
10. Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time.
11.Add 50 µL of 1x primary antibodies Anti-VAV1 (Phospho-Tyr174) Antibody, Anti-VAV1 Antibody and/or Anti-GAPDH Antibody) to the corresponding wells, cover with Parafilm and incubate for 16 hours (overnight) at 4°C. If the target expression is known to be high, incubate for 2 hours at room temperature.
12.Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time.
13.Add 50 µL of 1x secondary antibodies (HRP-Conjugated AntiRabbit IgG Antibody or HRP-Conjugated Anti-Mouse IgG Antibody) to corresponding wells and incubate for 1.5 hours at room temperature.
14.Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time.
15.Add 50 µL of Ready-to-Use Substrate to each well and incubate for 30 minutes at room temperature in the dark.
16.Add 50 µL of Stop Solution to each well and read OD at 450 nm immediately using the microplate reader.

(Additional Crystal Violet staining may be performed if desired – details of this may be found in the kit technical manual.)

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