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FLI1 Colorimetric Cell-Based ELISA Kit

SKU:
CBCAB00303
Product Type:
ELISA Kit
ELISA Type:
Cell Based
Research Area:
Epigenetics and Nuclear Signaling
Reactivity:
Human
Mouse
Detection Method:
Colorimetric
€599
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Description

system_update_altDatasheetMSDS

FLI1 Colorimetric Cell-Based ELISA Kit

The FLI1 Colorimetric Cell-Based ELISA Kit is a powerful tool for researchers looking to study the FLI1 protein in a cell-based system. This kit offers high sensitivity and specificity, allowing for accurate detection of FLI1 levels in cell samples.FLI1 is a transcription factor that plays a critical role in regulating gene expression and controlling cell growth and differentiation. Dysregulation of FLI1 has been linked to various diseases, including certain types of leukemia and autoimmune disorders, making it an important target for research and drug development.

With this ELISA kit, researchers can easily measure FLI1 levels in cell culture supernatants, providing valuable insights into the role of FLI1 in various cellular processes. The kit is easy to use and offers reliable and reproducible results, making it a valuable tool for studying FLI1 in cell-based models.

Product Name:FLI1 Colorimetric Cell-Based ELISA
Product Code:CBCAB00303
ELISA Type:Cell-Based
Target:FLI1
Reactivity:Human, Mouse
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:96-Well Microplate

The FLI1 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect FLI1 protein expression profile in cells. The kit can be used for measuring the relative amounts of FLI1 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on FLI1.

Qualitative determination of FLI1 concentration is achieved by an indirect ELISA format. In essence, FLI1 is captured by FLI1-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: 2313, UniProt ID: Q01543, OMIM: 193067, Unigene: Hs.504281
Gene Symbol:FLI1
Sub Type:None
UniProt Protein Function:FLI1: Sequence-specific transcriptional activator. Recognizes the DNA sequence 5'-C[CA]GGAAGT-3'. Defects in FLI1 are a cause of Ewing sarcoma (ES). A highly malignant, metastatic, primitive small round cell tumor of bone and soft tissue that affects children and adolescents. It belongs to the Ewing sarcoma family of tumors, a group of morphologically heterogeneous neoplasms that share the same cytogenetic features. They are considered neural tumors derived from cells of the neural crest. Ewing sarcoma represents the less differentiated form of the tumors. A chromosomal aberration involving FLI1 is found in patients with Erwing sarcoma. Translocation t(11;22)(q24;q12) with EWSR1. Belongs to the ETS family. 2 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:

Protein type:Transcription factor; Oncoprotein; DNA-binding

Chromosomal Location of Human Ortholog: 11q24.1-q24.3

Cellular Component: nucleus

Molecular Function:DNA binding; chromatin binding; transcription factor activity

Biological Process: regulation of transcription from RNA polymerase II promoter; transcription from RNA polymerase II promoter; organ morphogenesis; hemostasis; blood circulation; positive regulation of transcription from RNA polymerase II promoter; cell differentiation

NCBI Summary:This gene encodes a transcription factor containing an ETS DNA-binding domain. The gene can undergo a t(11;22)(q24;q12) translocation with the Ewing sarcoma gene on chromosome 22, which results in a fusion gene that is present in the majority of Ewing sarcoma cases. An acute lymphoblastic leukemia-associated t(4;11)(q21;q23) translocation involving this gene has also been identified. Alternative splicing results in multiple transcript variants. [provided by RefSeq, Aug 2012]
UniProt Code:Q01543
NCBI GenInfo Identifier:399496
NCBI Gene ID:2313
NCBI Accession:Q01543.1
UniProt Secondary Accession:Q01543,Q14319, Q92480, Q9UE07, B2R8H2, B4DFV4, B4DTC6 G3V183,
UniProt Related Accession:Q01543
Molecular Weight:
NCBI Full Name:Friend leukemia integration 1 transcription factor
NCBI Synonym Full Names:Fli-1 proto-oncogene, ETS transcription factor
NCBI Official Symbol:FLI1  
NCBI Official Synonym Symbols:EWSR2; SIC-1  
NCBI Protein Information:Friend leukemia integration 1 transcription factor; transcription factor ERGB; Ewing sarcoma breakpoint region 2; Friend leukemia virus integration 1
UniProt Protein Name:Friend leukemia integration 1 transcription factor
UniProt Synonym Protein Names:Proto-oncogene Fli-1; Transcription factor ERGB
Protein Family:Friend leukemia integration 1 transcription factor
UniProt Gene Name:FLI1  
UniProt Entry Name:FLI1_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-FLI1 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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