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FIR Colorimetric Cell-Based ELISA (CBCAB00958)

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SKU:
CBCAB00958
Product Type:
ELISA Kit
ELISA Type:
Cell Based
Reactivity:
Human
Mouse
Detection Method:
Colorimetric
€769

Description

system_update_altDatasheetMSDS

FIR Colorimetric Cell-Based ELISA

The FIR Colorimetric Cell-Based ELISA kit offered by AssayGenie is a cutting-edge tool for researchers to quantitatively measure the level of angiogenin in human samples, including serum, plasma, and cell culture supernatants. This kit is highly sensitive and specific, ensuring accurate and reproducible results for a variety of research applications.Angiogenin is a key player in angiogenesis, the process of new blood vessel formation, and is implicated in numerous diseases such as cancer, cardiovascular disorders, and neurodegenerative conditions.

By measuring angiogenin levels, researchers can gain valuable insights into disease mechanisms and potentially develop new therapeutic strategies.With AssayGenie's FIR Colorimetric Cell-Based ELISA kit, researchers can confidently study the role of angiogenin in various biological processes and disease states, advancing our understanding of angiogenesis and its implications for human health.

Product Name:FIR Colorimetric Cell-Based ELISA
Product Code:CBCAB00958
ELISA Type:Cell-Based
Target:FIR
Reactivity:Human, Mouse
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:96-Well Microplate

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

Qualitative determination of FIR concentration is achieved by an indirect ELISA format. In essence, FIR is captured by FIR-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: 9855, UniProt ID: O94887, OMIM: 0, Unigene: Hs.657318/Hs.679161
Gene Symbol:FARP2
Sub Type:None
UniProt Protein Function:FARP2: Rho-guanine nucleotide exchange factor that activates RAC1. Plays a role in the response to class 3 semaphorins and remodeling of the actin cytoskeleton. 2 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:

Protein type:Cell development/differentiation; GEF; GEF, Rac/Rho; Motility/polarity/chemotaxis

Chromosomal Location of Human Ortholog: 2q37.3

Cellular Component: cytoplasm; cytoskeleton; cytosol

Molecular Function:cytoskeletal protein binding; Rac guanyl-nucleotide exchange factor activity; Rho guanyl-nucleotide exchange factor activity

Biological Process: actin cytoskeleton reorganization; cell adhesion; hair cycle process; neuron remodeling; osteoclast differentiation; positive regulation of GTPase activity; Rac protein signal transduction; regulation of integrin activation; regulation of Rho protein signal transduction

UniProt Code:O94887
NCBI GenInfo Identifier:545478372
NCBI Gene ID:9855
NCBI Accession:NP_001269912.1
UniProt Secondary Accession:O94887,Q53QM5, Q8WU27, Q9UFE7, B7Z6J8, F5GZ84,
UniProt Related Accession:O94887
Molecular Weight:119 kDa
NCBI Full Name:FERM, ARHGEF and pleckstrin domain-containing protein 2 isoform b
NCBI Synonym Full Names:FERM, ARH/RhoGEF and pleckstrin domain protein 2
NCBI Official Symbol:FARP2  
NCBI Official Synonym Symbols:FIR; FRG; PLEKHC3  
NCBI Protein Information:FERM, ARHGEF and pleckstrin domain-containing protein 2; FERM, RhoGEF and pleckstrin domain-containing protein 2
UniProt Protein Name:FERM, ARHGEF and pleckstrin domain-containing protein 2
UniProt Synonym Protein Names:FERM domain-including RhoGEF; FIR; FERM, RhoGEF and pleckstrin domain-containing protein 2; Pleckstrin homology domain-containing family C member 3; PH domain-containing family C member 3
UniProt Gene Name:FARP2  
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-FIR 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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