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Keratin 5 Colorimetric Cell-Based ELISA Kit

SKU:
CBCAB00728
Product Type:
ELISA Kit
ELISA Type:
Cell Based
Reactivity:
Human
Mouse
Rat
Detection Method:
Colorimetric
$719
Frequently bought together:

Description

system_update_altDatasheetMSDS

Keratin 5 Colorimetric Cell-Based ELISA Kit

The Keratin 5 Colorimetric Cell-Based ELISA Kit is a cutting-edge tool designed for the precise measurement of Keratin 5 levels in cell lysates and culture supernatants. With its high sensitivity and specificity, this kit ensures accurate and reproducible results, making it perfect for various research applications.Keratin 5 is a key protein in the cytoskeleton of epithelial cells, playing a crucial role in maintaining cell integrity and providing structural support.

Abnormalities in Keratin 5 expression have been linked to various skin disorders, making it a valuable biomarker for studying skin health and diseases.Overall, the Keratin 5 Colorimetric Cell-Based ELISA Kit is a must-have for researchers looking to delve deeper into the role of Keratin 5 in cell biology and dermatology, opening up new avenues for potential therapeutic interventions. Visit the provided URL for more information and to order your kit today.

Product Name:Keratin 5 Colorimetric Cell-Based ELISA
Product Code:CBCAB00728
ELISA Type:Cell-Based
Target:Keratin 5
Reactivity:Human, Mouse, Rat
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:96-Well Microplate

The Keratin 5 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect Keratin 5 protein expression profile in cells. The kit can be used for measuring the relative amounts of Keratin 5 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on Keratin 5.

Qualitative determination of Keratin 5 concentration is achieved by an indirect ELISA format. In essence, Keratin 5 is captured by Keratin 5-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: 3852, UniProt ID: P13647, OMIM: 131760/131800/131900/131960/148040/179850/609352, Unigene: Hs.433845
Gene Symbol:KRT5
Sub Type:None
UniProt Protein Function:K5: Heterotetramer of two type I and two type II keratins. Keratin-5 associates with keratin-14. Interacts with TCHP. Belongs to the intermediate filament family.
UniProt Protein Details:

Protein type:Cytoskeletal

Chromosomal Location of Human Ortholog: 12q13.13

Cellular Component: mitochondrion; membrane; cytoplasm; keratin filament; plasma membrane; intermediate filament; cytosol; nucleus

Molecular Function:protein binding; structural constituent of cytoskeleton

Biological Process: epidermis development; hemidesmosome assembly

Disease: Epidermolysis Bullosa Simplex, Localized; Epidermolysis Bullosa Simplex, Generalized; Dowling-degos Disease 1; Epidermolysis Bullosa Simplex With Migratory Circinate Erythema; Epidermolysis Bullosa Simplex, Autosomal Recessive 1; Epidermolysis Bullosa Simplex With Mottled Pigmentation; Epidermolysis Bullosa Simplex, Dowling-meara Type

NCBI Summary:The protein encoded by this gene is a member of the keratin gene family. The type II cytokeratins consist of basic or neutral proteins which are arranged in pairs of heterotypic keratin chains coexpressed during differentiation of simple and stratified epithelial tissues. This type II cytokeratin is specifically expressed in the basal layer of the epidermis with family member KRT14. Mutations in these genes have been associated with a complex of diseases termed epidermolysis bullosa simplex. The type II cytokeratins are clustered in a region of chromosome 12q12-q13. [provided by RefSeq, Jul 2008]
UniProt Code:P13647
NCBI GenInfo Identifier:143811411
NCBI Gene ID:3852
NCBI Accession:P13647.3
UniProt Secondary Accession:P13647,Q6PI71, Q6UBJ0, Q8TA91,
UniProt Related Accession:P13647
Molecular Weight:590
NCBI Full Name:Keratin, type II cytoskeletal 5
NCBI Synonym Full Names:keratin 5
NCBI Official Symbol:KRT5  
NCBI Official Synonym Symbols:K5; CK5; DDD; DDD1; EBS2; KRT5A  
NCBI Protein Information:keratin, type II cytoskeletal 5; CK-5; cytokeratin-5; 58 kda cytokeratin; type-II keratin Kb5; epidermolysis bullosa simplex 2 Dowling-Meara/Kobner/Weber-Cockayne types; keratin 5 (epidermolysis bullosa simplex, Dowling-Meara/Kobner/Weber-Cockayne types)
UniProt Protein Name:Keratin, type II cytoskeletal 5
UniProt Synonym Protein Names:58 kDa cytokeratin; Cytokeratin-5; CK-5; Keratin-5; K5; Type-II keratin Kb5
Protein Family:Keratin
UniProt Gene Name:KRT5  
UniProt Entry Name:K2C5_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 5 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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