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Keratin 14 Colorimetric Cell-Based ELISA

SKU:
CBCAB00914
Product Type:
ELISA Kit
ELISA Type:
Cell Based
Reactivity:
Human
Mouse
Rat
Detection Method:
Colorimetric
$719
Frequently bought together:

Description

system_update_altDatasheetMSDS

Keratin 14 Colorimetric Cell-Based ELISA

The Keratin 14 Colorimetric Cell-Based ELISA kit is specifically designed for the precise measurement of keratin 14 levels in cell lysates, tissue homogenates, and other biological samples. This kit offers unparalleled sensitivity and accuracy, ensuring reliable and reproducible results for a variety of research applications.Keratin 14 is a key protein that plays a critical role in maintaining the structural integrity of epithelial cells, particularly in tissues such as skin and hair follicles.

Dysregulation of keratin 14 expression has been linked to various skin disorders and diseases, making it a crucial biomarker for studying these conditions and developing potential treatments.Whether investigating keratin biology, studying skin diseases, or exploring regenerative medicine applications, the Keratin 14 Colorimetric Cell-Based ELISA kit is an essential tool for researchers seeking to better understand the role of keratin 14 in cellular function and disease pathology.

Product Name:Keratin 14 Colorimetric Cell-Based ELISA
Product Code:CBCAB00914
ELISA Type:Cell-Based
Target:Keratin 14
Reactivity:Human, Mouse, Rat
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:96-Well Microplate

The Keratin 14 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect Keratin 14 protein expression profile in cells. The kit can be used for measuring the relative amounts of Keratin 14 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on Keratin 14.

Qualitative determination of Keratin 14 concentration is achieved by an indirect ELISA format. In essence, Keratin 14 is captured by Keratin 14-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: 3861/3868/3872, UniProt ID: P02533/P08779, OMIM: 125595/131760/131800/131900/148066/161000/601001/144200/148067/167200/161300/600962/148069/167210/184500, Unigene: Hs.654380/Hs.655160/Hs.2785
Gene Symbol:KRT14
Sub Type:None
UniProt Protein Function:K14: 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. There are two types of cytoskeletal and microfibrillar keratin: type I (acidic; 40-55 kDa) [K9 to K20] and type II (neutral to basic; 56-70 kDa) [K1 to K8]. Both a basic and an acidic keratin are required for filament assembly. Generally associates with K5.
UniProt Protein Details:

Protein type:Cytoskeletal

Chromosomal Location of Human Ortholog: 17q21.2

Cellular Component: cytoplasm; keratin filament; intermediate filament; cytosol; nucleus

Molecular Function:protein binding; structural constituent of cytoskeleton

Biological Process: epidermis development; hemidesmosome assembly; epithelial cell differentiation; response to zinc ion; response to ionizing radiation; intermediate filament bundle assembly; hair cycle; aging

Disease: Epidermolysis Bullosa Simplex, Localized; Epidermolysis Bullosa Simplex, Generalized; Dermatopathia Pigmentosa Reticularis; Epidermolysis Bullosa Simplex, Autosomal Recessive 1; Naegeli Syndrome; Epidermolysis Bullosa Simplex, Dowling-meara Type

NCBI Summary:This gene encodes a member of the keratin family, the most diverse group of intermediate filaments. This gene product, a type I keratin, is usually found as a heterotetramer with two keratin 5 molecules, a type II keratin. Together they form the cytoskeleton of epithelial cells. Mutations in the genes for these keratins are associated with epidermolysis bullosa simplex. At least one pseudogene has been identified at 17p12-p11. [provided by RefSeq, Jul 2008]
UniProt Code:P02533
NCBI GenInfo Identifier:229463044
NCBI Gene ID:3861
NCBI Accession:P02533.4
UniProt Secondary Accession:P02533,P08779,
UniProt Related Accession:P02533
Molecular Weight:~ 52kDa
NCBI Full Name:Keratin, type I cytoskeletal 14
NCBI Synonym Full Names:keratin 14
NCBI Official Symbol:KRT14  
NCBI Official Synonym Symbols:K14; NFJ; CK14; EBS3; EBS4  
NCBI Protein Information:keratin, type I cytoskeletal 14
UniProt Protein Name:Keratin, type I cytoskeletal 14
UniProt Synonym Protein Names:Cytokeratin-14; CK-14; Keratin-14; K14
Protein Family:Putative outer capsid protein
UniProt Gene Name:KRT14  
UniProt Entry Name:K1C14_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 14 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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