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CBL (Phospho-Tyr700) Colorimetric Cell-Based ELISA Kit

SKU:
CBCAB01287
Product Type:
ELISA Kit
ELISA Type:
Cell Based Phospho Specific
Research Area:
Signal Transduction
Reactivity:
Human
Mouse
Rat
Detection Method:
Colorimetric
€599
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Description

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CBL (Phospho-Tyr700)Colorimetric Cell-Based ELISA Kit

The CBL (Cbl Proto-Oncogene) Phospho-Tyr700 Colorimetric Cell-Based ELISA Kit is a powerful tool for detecting phosphorylated CBL levels in cell lysates. This kit offers high sensitivity and specificity, providing accurate and reproducible results for a variety of research applications.CBL is a key regulator of cell signaling and plays a crucial role in various cellular processes, including growth, differentiation, and immune response. Phosphorylation of CBL at Tyr700 is known to modulate its function and is implicated in diseases such as cancer and autoimmune disorders.

With this ELISA kit, researchers can quickly and easily quantify phosphorylated CBL levels in cell samples, enabling a better understanding of its role in disease progression and potential therapeutic interventions. Order your CBL Phospho-Tyr700 Colorimetric Cell-Based ELISA Kit today and take your research to the next level.

Product Name:CBL (Phospho-Tyr700) Colorimetric Cell-Based ELISA
Product Code:CBCAB01287
ELISA Type:Cell-Based
Target:CBL (Phospho-Tyr700)
Reactivity:Human, Mouse, Rat
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nm
Format:2 x 96-Well Microplates

The CBL (Phospho-Tyr700) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect CBL protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated CBL in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on CBL phosphorylation.

Qualitative determination of CBL (Phospho-Tyr700) concentration is achieved by an indirect ELISA format. In essence, CBL (Phospho-Tyr700) is captured by CBL (Phospho-Tyr700)-specific primary (1ø) antibodies while the HRP-conjugated secondary (2ø) antibodies bind the Fc region of the 1ø antibody. Through this binding, the HRP enzyme conjugated to the 2ø 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: 867, UniProt ID: P22681, OMIM: 165360, Unigene: Hs.504096
Gene Symbol:CBL
Sub Type:Phospho
UniProt Protein Function:Adapter protein that functions as a negative regulator of many signaling pathways that are triggered by activation of cell surface receptors. Acts as an E3 ubiquitin-protein ligase, which accepts ubiquitin from specific E2 ubiquitin-conjugating enzymes, and then transfers it to substrates promoting their degradation by the proteasome. Recognizes activated receptor tyrosine kinases, including KIT, FLT1, FGFR1, FGFR2, PDGFRA, PDGFRB, EGFR, CSF1R, EPHA8 and KDR and terminates signaling. Recognizes membrane-bound HCK, SRC and other kinases of the SRC family and mediates their ubiquitination and degradation. Participates in signal transduction in hematopoietic cells. Plays an important role in the regulation of osteoblast differentiation and apoptosis. Essential for osteoclastic bone resorption. The 'Tyr-731' phosphorylated form induces the activation and recruitment of phosphatidylinositol 3-kinase to the cell membrane in a signaling pathway that is critical for osteoclast function. May be functionally coupled with the E2 ubiquitin-protein ligase UB2D3.
NCBI Summary:This gene is a proto-oncogene that encodes a RING finger E3 ubiquitin ligase. The encoded protein is one of the enzymes required for targeting substrates for degradation by the proteasome. This protein mediates the transfer of ubiquitin from ubiquitin conjugating enzymes (E2) to specific substrates. This protein also contains an N-terminal phosphotyrosine binding domain that allows it to interact with numerous tyrosine-phosphorylated substrates and target them for proteasome degradation. As such it functions as a negative regulator of many signal transduction pathways. This gene has been found to be mutated or translocated in many cancers including acute myeloid leukaemia, and expansion of CGG repeats in the 5' UTR has been associated with Jacobsen syndrome. Mutations in this gene are also the cause of Noonan syndrome-like disorder. [provided by RefSeq, Jul 2016]
UniProt Code:P22681
NCBI GenInfo Identifier:251757253
NCBI Gene ID:867
NCBI Accession:P22681.2
UniProt Secondary Accession:P22681,A3KMP8,
UniProt Related Accession:P22681
Molecular Weight:99,633 Da
NCBI Full Name:E3 ubiquitin-protein ligase CBL
NCBI Synonym Full Names:Cbl proto-oncogene
NCBI Official Symbol:CBL  
NCBI Official Synonym Symbols:CBL2; NSLL; C-CBL; RNF55; FRA11B  
NCBI Protein Information:E3 ubiquitin-protein ligase CBL
UniProt Protein Name:E3 ubiquitin-protein ligase CBL
UniProt Synonym Protein Names:Casitas B-lineage lymphoma proto-oncogene; Proto-oncogene c-Cbl; RING finger protein 55; RING-type E3 ubiquitin transferase CBLCurated; Signal transduction protein CBL
Protein Family:E3 ubiquitin ligase
UniProt Gene Name:CBL  
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-CBL (Phospho-Tyr700) Antibody, Anti-CBL 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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