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FRK Colorimetric Cell-Based ELISA (CBCAB01014)

SKU:
CBCAB01014
Product Type:
ELISA Kit
ELISA Type:
Cell Based
Reactivity:
Human
Mouse
Rat
Detection Method:
Colorimetric
€769

Description

system_update_altDatasheetMSDS

FRK Colorimetric Cell-Based ELISA

The FRK Colorimetric Cell-Based ELISA Kit is specifically designed for the detection of FRK (Fyn-related kinase) levels in cell lysates and culture supernatants. This kit offers high sensitivity and specificity, ensuring accurate and reproducible results for a variety of research applications.FRK is a key protein involved in cell signaling pathways, regulating processes such as cell growth, differentiation, and survival. Abnormalities in FRK expression and activity have been linked to various diseases, including cancer and neurodegenerative disorders, making it an important target for research and potential therapeutic development.

With the FRK Colorimetric Cell-Based ELISA Kit, researchers can confidently measure FRK levels and gain valuable insights into its role in cellular signaling and disease pathology. This kit provides a reliable tool for studying FRK function and potential therapeutic interventions.

Product Name:FRK Colorimetric Cell-Based ELISA
Product Code:CBCAB01014
ELISA Type:Cell-Based
Target:FRK
Reactivity:Human, Mouse, Rat
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:96-Well Microplate

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

Qualitative determination of FRK concentration is achieved by an indirect ELISA format. In essence, FRK is captured by FRK-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: 2444, UniProt ID: P42685, OMIM: 606573, Unigene: Hs.89426
Gene Symbol:FRK
Sub Type:None
UniProt Protein Function:FRK: a tyrosine kinase of the Src family. Expressed in cell lines derived from tissues of lymphoid, brain, breast, colon and bladder origin. A nuclear protein that may function during G1 and S phase of the cell cycle to suppress growth.
UniProt Protein Details:

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

Chromosomal Location of Human Ortholog: 6q21-q22.3

Cellular Component: cytoplasm; extrinsic to internal side of plasma membrane; intracellular; intracellular membrane-bound organelle; nucleus

Molecular Function:protein binding; receptor binding

Biological Process: cell differentiation; innate immune response; negative regulation of cell proliferation; negative regulation of transcription from RNA polymerase II promoter; protein amino acid phosphorylation; transmembrane receptor protein tyrosine kinase signaling pathway

NCBI Summary:The protein encoded by this gene belongs to the TYR family of protein kinases. This tyrosine kinase is a nuclear protein and may function during G1 and S phase of the cell cycle and suppress growth. [provided by RefSeq, Jul 2008]
UniProt Code:P42685
NCBI GenInfo Identifier:1169745
NCBI Gene ID:2444
NCBI Accession:P42685.1
UniProt Secondary Accession:P42685,Q13128, Q9NTR5, B4DY49,
UniProt Related Accession:P42685
Molecular Weight:41,980 Da
NCBI Full Name:Tyrosine-protein kinase FRK
NCBI Synonym Full Names:fyn related Src family tyrosine kinase
NCBI Official Symbol:FRK  
NCBI Official Synonym Symbols:GTK; RAK; PTK5  
NCBI Protein Information:tyrosine-protein kinase FRK
UniProt Protein Name:Tyrosine-protein kinase FRK
UniProt Synonym Protein Names:FYN-related kinase; Nuclear tyrosine protein kinase RAK; Protein-tyrosine kinase 5
Protein Family:Tyrosine-protein kinase
UniProt Gene Name:FRK  
UniProt Entry Name:FRK_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-FRK 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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