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FOXK1 Colorimetric Cell-Based ELISA

SKU:
CBCAB01055
Product Type:
ELISA Kit
ELISA Type:
Cell Based
Research Area:
Developmental Biology
Reactivity:
Human
Mouse
Detection Method:
Colorimetric
$719
Frequently bought together:

Description

system_update_altDatasheetMSDS

FOXK1 Colorimetric Cell-Based ELISA

The FOXK1 Colorimetric Cell-Based ELISA Kit from Assay Genie is specifically designed for the detection of Forkhead box K1 (FOXK1) levels in cell lysates. This kit offers high sensitivity and accuracy, allowing for precise measurement of FOXK1 levels in a variety of cell types and conditions.FOXK1 is a transcription factor that plays a crucial role in various cellular processes, including cell cycle regulation, DNA damage response, and apoptosis. Dysregulation of FOXK1 has been implicated in cancer progression and other diseases, making it a valuable target for research and drug development.

With the FOXK1 Colorimetric Cell-Based ELISA Kit, researchers can easily and accurately quantify FOXK1 levels in their cell samples, providing valuable insights into the role of FOXK1 in disease pathogenesis and potential therapeutic interventions. Trust Assay Genie for reliable and innovative tools for your research needs.

Product Name:FOXK1 Colorimetric Cell-Based ELISA
Product Code:CBCAB01055
ELISA Type:Cell-Based
Target:FOXK1
Reactivity:Human, Mouse
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:96-Well Microplate

The FOXK1 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect FOXK1 protein expression profile in cells. The kit can be used for measuring the relative amounts of FOXK1 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on FOXK1.

Qualitative determination of FOXK1 concentration is achieved by an indirect ELISA format. In essence, FOXK1 is captured by FOXK1-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: 221937, UniProt ID: P85037, OMIM: 0, Unigene: Hs.487393
Gene Symbol:FOXK1
Sub Type:None
UniProt Protein Function:FOXK1: Transcriptional regulator that binds to the upstream enhancer region (CCAC box) of myoglobin gene. Has a role in myogenic differentiation and in remodeling processes of adult muscles that occur in response to physiological stimuli. 2 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:

Protein type:Transcription factor; DNA-binding

Chromosomal Location of Human Ortholog: 7p22.1

Cellular Component: nucleus

Molecular Function:protein binding

Biological Process: anatomical structure morphogenesis; cell differentiation; muscle development; negative regulation of cell growth; negative regulation of transcription, DNA-dependent; positive regulation of transcription, DNA-dependent; regulation of transcription from RNA polymerase II promoter; transcription, DNA-dependent

UniProt Code:P85037
NCBI GenInfo Identifier:118572324
NCBI Gene ID:221937
NCBI Accession:P85037.1
UniProt Secondary Accession:P85037,P42128,
UniProt Related Accession:P85037
Molecular Weight:59,702 Da
NCBI Full Name:Forkhead box protein K1
NCBI Synonym Full Names:forkhead box K1
NCBI Official Symbol:FOXK1  
NCBI Official Synonym Symbols:FOXK1L  
NCBI Protein Information:forkhead box protein K1
UniProt Protein Name:Forkhead box protein K1
UniProt Synonym Protein Names:Myocyte nuclear factor; MNF
Protein Family:Forkhead box protein
UniProt Gene Name:FOXK1  
UniProt Entry Name:FOXK1_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-FOXK1 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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