FAKD3 Colorimetric Cell-Based ELISA

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SKU:
CBCAB01102
Product Type:
ELISA Kit
ELISA Type:
Cell Based
Reactivity:
Human
Detection Method:
Colorimetric
Frequently bought together:

Description

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FAKD3 Colorimetric Cell-Based ELISA

The Human FAKD3 Colorimetric Cell-Based ELISA Kit is a cutting-edge tool for the accurate detection of FAKD3 levels in cell cultures. This kit is designed with high sensitivity and specificity, ensuring precise and reliable results for a wide range of research applications.FAKD3 is a critical protein involved in cell adhesion and migration, playing a key role in various cellular processes. Understanding FAKD3 levels can provide valuable insights into cell signaling pathways, cancer progression, and potential therapeutic targets.

The easy-to-use colorimetric assay allows for quick and efficient quantification of FAKD3 levels in cell-based experiments, enabling researchers to study the impact of this protein on cellular functions and disease pathways. Trust the Human FAKD3 Colorimetric Cell-Based ELISA Kit for accurate and reproducible results in your research endeavors.

Product Name:FAKD3 Colorimetric Cell-Based ELISA
Product Code:CBCAB01102
ELISA Type:Cell-Based
Target:FAKD3
Reactivity:Human
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:96-Well Microplate

The FAKD3 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect FAKD3 protein expression profile in cells. The kit can be used for measuring the relative amounts of FAKD3 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on FAKD3.

Qualitative determination of FAKD3 concentration is achieved by an indirect ELISA format. In essence, FAKD3 is captured by FAKD3-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: 79072, UniProt ID: Q14CZ7, OMIM: 0, Unigene: Hs.653162
Gene Symbol:FAKD3
Sub Type:None
UniProt Protein Function:FASTKD3: Belongs to the FAST kinase family.
UniProt Protein Details:

Chromosomal Location of Human Ortholog: 5p15.31

Cellular Component: mitochondrion

Molecular Function:protein binding

Biological Process: cellular respiration

NCBI Summary:This gene encodes a member of a small family of Fas-activated serine/threonine kinase domain (FASTKD) containing proteins that share an amino terminal mitochondrial targeting domain and multiple carboxy terminal FAST domains as well as a putative RNA-binding RAP domain. The members of this family are ubiquitously expressed and are generally most abundant in mitochondria-enriched tissues such as heart, skeletal muscle and brown-adipose tissue. Some members of this protein family may play a role in apoptosis. The protein encoded by this gene interacts with components of the mitochondrial respiratory and translation networks. A pseudogene of this gene is also present on chromosome 5. Alternative splicing results in multiple transcript variants. [provided by RefSeq, Jan 2013]
UniProt Code:Q14CZ7
NCBI GenInfo Identifier:294862434
NCBI Gene ID:79072
NCBI Accession:Q14CZ7.2
UniProt Secondary Accession:Q14CZ7,Q9BVD3,
UniProt Related Accession:Q14CZ7
Molecular Weight:75,689 Da
NCBI Full Name:FAST kinase domain-containing protein 3, mitochondrial
NCBI Synonym Full Names:FAST kinase domains 3
NCBI Official Symbol:FASTKD3  
NCBI Protein Information:FAST kinase domain-containing protein 3, mitochondrial
UniProt Protein Name:FAST kinase domain-containing protein 3, mitochondrial
Protein Family:FAST kinase domain-containing protein
UniProt Gene Name:FASTKD3  
UniProt Entry Name:FAKD3_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-FAKD3 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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