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WASP Colorimetric Cell-Based ELISA Kit

SKU:
CBCAB00903
Product Type:
ELISA Kit
ELISA Type:
Cell Based
Reactivity:
Human
Mouse
Rat
Detection Method:
Colorimetric
€599
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Description

system_update_altDatasheetMSDS

WASP Colorimetric Cell-Based ELISA Kit

The WASP Colorimetric Cell-Based ELISA Kit is a cutting-edge tool designed for the precise measurement of WASP (Wiskott-Aldrich Syndrome Protein) levels in cell lysates and tissue homogenates. This kit offers exceptional sensitivity and specificity, ensuring accurate and consistent results for a variety of research applications.WASP is a key regulator of actin cytoskeleton dynamics, playing a crucial role in cell migration, adhesion, and signaling. Dysregulation of WASP has been linked to various immune disorders, including Wiskott-Aldrich Syndrome, autoimmune diseases, and cancer.

By accurately quantifying WASP levels, researchers can gain valuable insights into the molecular mechanisms underlying these diseases and potentially identify new therapeutic targets.Overall, the WASP Colorimetric Cell-Based ELISA Kit provides researchers with a powerful tool to advance their studies in cell biology, immunology, and cancer research, ultimately contributing to the development of novel treatment strategies for a range of diseases.

Product Name:WASP Colorimetric Cell-Based ELISA
Product Code:CBCAB00903
ELISA Type:Cell-Based
Target:WASP
Reactivity:Human, Mouse, Rat
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:96-Well Microplate

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

Qualitative determination of WASP concentration is achieved by an indirect ELISA format. In essence, WASP is captured by WASP-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: 7454, UniProt ID: P42768, OMIM: 300299/300392/301000/313900, Unigene: Hs.2157
Gene Symbol:WAS
Sub Type:None
UniProt Protein Function:WASP: a member of the Wiskott-Aldrich syndrome (WAS) family of proteins. A cytoplasmic protein expressed exclusively in hematopoietic cells. Transduces signals from surface receptors to the actin cytoskeleton. Associates with the small GTPase, Cdc42, known to regulate formation of actin filaments, and the cytoskeletal organizing complex, Arp2/3. Mutated in Wiskott-Aldrich syndrome, a rare, inherited, X-linked, recessive disease characterized by immune dysregulation and microthrombocytopenia.
UniProt Protein Details:

Protein type:Motility/polarity/chemotaxis; Adaptor/scaffold

Chromosomal Location of Human Ortholog: Xp11.4-p11.21

Cellular Component: intercellular junction; vesicle membrane; cytosol; actin cytoskeleton

Molecular Function:identical protein binding; protein binding; phospholipase binding; actin binding; SH3 domain binding; protein kinase binding

Biological Process: epidermis development; T cell activation; actin filament polymerization; regulation of catalytic activity; actin filament-based movement; actin polymerization and/or depolymerization; innate immune response; defense response; protein complex assembly; immune response; endosome transport; blood coagulation; T cell receptor signaling pathway

Disease: Thrombocytopenia 1; Neutropenia, Severe Congenital, X-linked

NCBI Summary:The Wiskott-Aldrich syndrome (WAS) family of proteins share similar domain structure, and are involved in transduction of signals from receptors on the cell surface to the actin cytoskeleton. The presence of a number of different motifs suggests that they are regulated by a number of different stimuli, and interact with multiple proteins. Recent studies have demonstrated that these proteins, directly or indirectly, associate with the small GTPase, Cdc42, known to regulate formation of actin filaments, and the cytoskeletal organizing complex, Arp2/3. Wiskott-Aldrich syndrome is a rare, inherited, X-linked, recessive disease characterized by immune dysregulation and microthrombocytopenia, and is caused by mutations in the WAS gene. The WAS gene product is a cytoplasmic protein, expressed exclusively in hematopoietic cells, which show signalling and cytoskeletal abnormalities in WAS patients. A transcript variant arising as a result of alternative promoter usage, and containing a different 5' UTR sequence, has been described, however, its full-length nature is not known. [provided by RefSeq, Jul 2008]
UniProt Code:P42768
NCBI GenInfo Identifier:1722836
NCBI Gene ID:7454
NCBI Accession:P42768.4
UniProt Secondary Accession:P42768,Q9BU11, Q9UNJ9,
UniProt Related Accession:P42768
Molecular Weight:502
NCBI Full Name:Wiskott-Aldrich syndrome protein
NCBI Synonym Full Names:Wiskott-Aldrich syndrome
NCBI Official Symbol:WAS  
NCBI Official Synonym Symbols:THC; IMD2; SCNX; THC1; WASP  
NCBI Protein Information:wiskott-Aldrich syndrome protein; eczema-thrombocytopenia; thrombocytopenia 1 (X-linked)
UniProt Protein Name:Wiskott-Aldrich syndrome protein
Protein Family:Waspkinin
UniProt Gene Name:WAS  
UniProt Entry Name:WASP_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-WASP 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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