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BTK Colorimetric Cell-Based ELISA Kit (CBCAB00114)

SKU:
CBCAB00114
Product Type:
ELISA Kit
ELISA Type:
Cell Based
Research Area:
Immunology
Reactivity:
Human
Mouse
Rat
Detection Method:
Colorimetric
€769

Description

system_update_altDatasheetMSDS

BTK Colorimetric Cell-Based ELISA Kit

The BTK Colorimetric Cell-Based ELISA Kit is a cutting-edge tool designed for the precise measurement of Bruton's tyrosine kinase (BTK) activity in cell lysates. With its superior sensitivity and specificity, this kit delivers consistent and accurate results, making it an invaluable asset for researchers across various fields.BTK is a key regulator of B cell development and function, playing a critical role in immune response and autoimmune diseases. Dysregulation of BTK activity has been implicated in conditions such as leukemia, lymphoma, and rheumatoid arthritis, highlighting its importance as a therapeutic target and diagnostic marker.

By utilizing the BTK Colorimetric Cell-Based ELISA Kit, researchers can unravel the complex mechanisms underlying BTK signaling pathways, paving the way for innovative discoveries and potential therapeutic interventions. Elevate your research workflow with this advanced kit and unlock new insights into BTK biology.

Product Name:BTK Colorimetric Cell-Based ELISA
Product Code:CBCAB00114
ELISA Type:Cell-Based
Target:BTK
Reactivity:Human, Mouse, Rat
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:96-Well Microplate

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

Qualitative determination of BTK concentration is achieved by an indirect ELISA format. In essence, BTK is captured by BTK-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: 695, UniProt ID: Q06187, OMIM: 300300/300755/307200, Unigene: Hs.159494
Gene Symbol:BTK
Sub Type:None
UniProt Protein Function:Btk: a tyrosine kinase of the Tec family. Plays a crucial role in B-cell ontogeny. Defects cause X-linked agammaglobulinemia, an immunodeficiency characterized by failure to produce mature B lymphocyte cells and associated with a failure of Ig heavy chain rearrangement. Truncated splice forms found in childhood leukemias may underlie radiation resistance of tumors through inhibition of apoptosis.
UniProt Protein Details:

Protein type:Kinase, protein; Protein kinase, TK; EC 2.7.10.2; Protein kinase, tyrosine (non-receptor); TK group; Tec family

Cellular Component: cytoplasm; cytoplasmic vesicle; cytosol; extrinsic to internal side of plasma membrane; intracellular membrane-bound organelle; lipid raft; mast cell granule; membrane; nucleus; perinuclear region of cytoplasm; plasma membrane

Molecular Function:ATP binding; identical protein binding; kinase activity; lipid binding; metal ion binding; non-membrane spanning protein tyrosine kinase activity; nucleotide binding; phosphatidylinositol-3,4,5-triphosphate binding; protein binding; protein kinase activity; protein-tyrosine kinase activity; transferase activity

Biological Process: adaptive immune response; apoptosis; cell maturation; histamine secretion by mast cell; I-kappaB kinase/NF-kappaB cascade; immune system process; innate immune response; negative regulation of cytokine production; peptidyl-tyrosine phosphorylation; phosphorylation; protein amino acid autophosphorylation; protein amino acid phosphorylation; regulation of cell proliferation; regulation of transcription, DNA-dependent; response to organic substance; transcription, DNA-dependent; transmembrane receptor protein tyrosine kinase signaling pathway

NCBI Summary:The protein encoded by this gene plays a crucial role in B-cell development. Mutations in this gene cause X-linked agammaglobulinemia type 1, which is an immunodeficiency characterized by the failure to produce mature B lymphocytes, and associated with a failure of Ig heavy chain rearrangement. Alternative splicing results in multiple transcript variants encoding different isoforms. [provided by RefSeq, Dec 2013]
UniProt Code:Q06187
NCBI GenInfo Identifier:547759
NCBI Gene ID:695
NCBI Accession:Q06187.3
UniProt Secondary Accession:Q06187,Q61365,
UniProt Related Accession:Q06187
Molecular Weight:
NCBI Full Name:Tyrosine-protein kinase BTK
NCBI Synonym Full Names:Bruton tyrosine kinase
NCBI Official Symbol:BTK  
NCBI Official Synonym Symbols:AT; ATK; BPK; XLA; IMD1; AGMX1; PSCTK1  
NCBI Protein Information:tyrosine-protein kinase BTK
UniProt Protein Name:Tyrosine-protein kinase BTK
UniProt Synonym Protein Names:Agammaglobulinemia tyrosine kinase; ATK; B-cell progenitor kinase; BPK; Bruton tyrosine kinase; Kinase EMB
Protein Family:Tyrosine-protein kinase
UniProt Gene Name:Btk  
UniProt Entry Name:BTK_MOUSE
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-BTK 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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