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Keratin 18 (Phospho-Ser52) Colorimetric Cell-Based ELISA Kit

SKU:
CBCAB01408
Product Type:
ELISA Kit
ELISA Type:
Cell Based Phospho Specific
Research Area:
Cell Cycle
Reactivity:
Human
Detection Method:
Colorimetric
€599
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Description

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Keratin 18 (Phospho-Ser52)Colorimetric Cell-Based ELISA Kit

The Keratin 18 (Phospho-Ser52) Colorimetric Cell-Based ELISA Kit is specifically designed for the precise measurement of phospho-ser52 keratin 18 levels in cell lysates. This kit offers high sensitivity and accuracy, ensuring consistent and trustworthy results for various research applications.Keratin 18 is a key protein involved in maintaining the structural integrity of epithelial cells, and phosphorylation at serine 52 is known to modulate its function. Dysregulation of keratin 18 phosphorylation has been linked to various pathological conditions such as liver disease, cancer, and inflammatory disorders, making it a valuable biomarker for understanding these diseases and exploring potential therapeutic options.

The Keratin 18 (Phospho-Ser52) Colorimetric Cell-Based ELISA Kit provides researchers with a reliable tool to investigate the role of keratin 18 phosphorylation in disease pathogenesis and treatment strategies. Its user-friendly format and high-performance characteristics make it an essential component for studies in cell biology and molecular medicine.

Product Name:Keratin 18 (Phospho-Ser52) Colorimetric Cell-Based ELISA
Product Code:CBCAB01408
ELISA Type:Cell-Based
Target:Keratin 18 (Phospho-Ser52)
Reactivity:Human
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nm
Format:2 x 96-Well Microplates

The Keratin 18 (Phospho-Ser52) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect Keratin 18 protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated Keratin 18 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on Keratin 18 phosphorylation.

Qualitative determination of Keratin 18 (Phospho-Ser52) concentration is achieved by an indirect ELISA format. In essence, Keratin 18 (Phospho-Ser52) is captured by Keratin 18 (Phospho-Ser52)-specific primary (1ø) antibodies while the HRP-conjugated secondary (2ø) antibodies bind the Fc region of the 1ø antibody. Through this binding, the HRP enzyme conjugated to the 2ø 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: 3875, UniProt ID: P05783, OMIM: 148070/215600, Unigene: Hs.406013
Gene Symbol:KRT18
Sub Type:Phospho
UniProt Protein Function:K18: a type I cytoskeletal keratin. The keratins are intermediate filament proteins responsible for the structural integrity of epithelial cells and are subdivided into cytokeratins and hair keratins. Keratin 18 and its filament partner keratin 8 are perhaps the most commonly found members of the intermediate filament gene family. They are expressed in single layer epithelial tissues of the body. Defects are a cause of cryptogenic cirrhosis.
UniProt Protein Details:

Protein type:Nucleolus; Cytoskeletal

Chromosomal Location of Human Ortholog: 12q13

Cellular Component: cytoplasm; intermediate filament; keratin filament; microtubule organizing center; nucleolus; perinuclear region of cytoplasm

Molecular Function:protein binding; structural molecule activity

Biological Process: anatomical structure morphogenesis; cell cycle; Golgi to plasma membrane CFTR protein transport; intermediate filament cytoskeleton organization and biogenesis; negative regulation of apoptosis; tumor necrosis factor-mediated signaling pathway; viral reproduction

Disease: Cirrhosis, Familial

NCBI Summary:KRT18 encodes the type I intermediate filament chain keratin 18. Keratin 18, together with its filament partner keratin 8, are perhaps the most commonly found members of the intermediate filament gene family. They are expressed in single layer epithelial tissues of the body. Mutations in this gene have been linked to cryptogenic cirrhosis. Two transcript variants encoding the same protein have been found for this gene. [provided by RefSeq, Jul 2008]
UniProt Code:P05783
NCBI GenInfo Identifier:125083
NCBI Gene ID:3875
NCBI Accession:P05783.2
UniProt Secondary Accession:P05783,Q53G38, Q5U0N8, Q9BW26,
UniProt Related Accession:P05783
Molecular Weight:
NCBI Full Name:Keratin, type I cytoskeletal 18
NCBI Synonym Full Names:keratin 18
NCBI Official Symbol:KRT18  
NCBI Official Synonym Symbols:K18; CK-18; CYK18  
NCBI Protein Information:keratin, type I cytoskeletal 18
UniProt Protein Name:Keratin, type I cytoskeletal 18
UniProt Synonym Protein Names:Cell proliferation-inducing gene 46 protein; Cytokeratin-18; CK-18; Keratin-18; K18
Protein Family:Keratin
UniProt Gene Name:KRT18  
UniProt Entry Name:K1C18_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-Keratin 18 (Phospho-Ser52) Antibody, Anti-Keratin 18 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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