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Keratin 19 Colorimetric Cell-Based ELISA Kit

SKU:
CBCAB00726
Product Type:
ELISA Kit
ELISA Type:
Cell Based
Research Area:
Immunology
Reactivity:
Human
Mouse
Rat
Detection Method:
Colorimetric
€599
Frequently bought together:

Description

system_update_altDatasheetMSDS

Keratin 19 Colorimetric Cell-Based ELISA Kit

The Keratin 19 Colorimetric Cell-Based ELISA Kit from Assay Genie is a cutting-edge tool for detecting and quantifying levels of Keratin 19, a key protein involved in the structural integrity of epithelial cells. This kit is optimized for use in cell-based assays, providing accurate and reliable results for researchers studying cell biology, cancer, and dermatology.Keratin 19 is known to be overexpressed in various cancers, including breast, liver, and pancreatic cancer, making it a valuable biomarker for early detection and monitoring of these diseases.

By measuring Keratin 19 levels with precision and sensitivity, researchers can gain valuable insights into disease progression and potential therapeutic targets.The Assay Genie Keratin 19 Colorimetric Cell-Based ELISA Kit offers high performance and ease of use, making it an essential tool for understanding the role of Keratin 19 in health and disease. Whether studying cell differentiation, tissue regeneration, or cancer progression, this kit provides the accuracy and consistency needed for successful research outcomes.

Product Name:Keratin 19 Colorimetric Cell-Based ELISA
Product Code:CBCAB00726
ELISA Type:Cell-Based
Target:Keratin 19
Reactivity:Human, Mouse, Rat
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:96-Well Microplate

The Keratin 19 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect Keratin 19 protein expression profile in cells. The kit can be used for measuring the relative amounts of Keratin 19 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on Keratin 19.

Qualitative determination of Keratin 19 concentration is achieved by an indirect ELISA format. In essence, Keratin 19 is captured by Keratin 19-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: 3880, UniProt ID: P08727, OMIM: 148020, Unigene: Hs.654568
Gene Symbol:KRT19
Sub Type:None
UniProt Protein Function:Function: Involved in the organization of myofibers. Together with KRT8, helps to link the contractile apparatus to dystrophin at the costameres of striated muscle. Ref.14
UniProt Protein Details:

Subunit structure: Heterotetramer of two type I and two type II keratins. Interacts with PNN and the actin-binding domain of DMD. Interacts with HCV core protein. Ref.13 Ref.14

Tissue specificity: Expressed in a defined zone of basal keratinocytes in the deep outer root sheath of hair follicles. Also observed in sweat gland and mammary gland ductal and secretory cells, bile ducts, gastrointestinal tract, bladder urothelium, oral epithelia, esophagus, ectocervical epithelium (at protein level). Expressed in epidermal basal cells, in nipple epidermis and a defined region of the hair follicle. Also seen in a subset of vascular wall cells in both the veins and artery of human umbilical cord, and in umbilical cord vascular smooth muscle. Observed in muscle fibers accumulating in the costameres of myoplasm at the sarcolemma in structures that contain dystrophin and spectrin. Ref.2 Ref.4 Ref.14

Developmental stage: Present in hair follicles at all stages of development. Ref.4

Domain: This keratin differs from all other IF proteins in lacking the C-terminal tail domain.

Miscellaneous: There are two types of cytoskeletal and microfibrillar keratin: I (acidic; 40-55 kDa) and II (neutral to basic; 56-70 kDa).

Sequence similarities: Belongs to the intermediate filament family.

NCBI Summary:The protein encoded by this gene is a member of the keratin family. The keratins are intermediate filament proteins responsible for the structural integrity of epithelial cells and are subdivided into cytokeratins and hair keratins. The type I cytokeratins consist of acidic proteins which are arranged in pairs of heterotypic keratin chains. Unlike its related family members, this smallest known acidic cytokeratin is not paired with a basic cytokeratin in epithelial cells. It is specifically expressed in the periderm, the transiently superficial layer that envelopes the developing epidermis. The type I cytokeratins are clustered in a region of chromosome 17q12-q21. [provided by RefSeq, Jul 2008]
UniProt Code:P08727
NCBI GenInfo Identifier:311033484
NCBI Gene ID:3880
NCBI Accession:P08727.4
UniProt Secondary Accession:P08727,Q5XG83, Q6NW33, Q7L5M9, Q96A53, Q96FV1, Q9BYF9 Q9P1Y4, B2R874,
UniProt Related Accession:P08727
Molecular Weight:44,106 Da
NCBI Full Name:Keratin, type I cytoskeletal 19
NCBI Synonym Full Names:keratin 19
NCBI Official Symbol:KRT19  
NCBI Official Synonym Symbols:K19; CK19; K1CS  
NCBI Protein Information:keratin, type I cytoskeletal 19; CK-19; keratin-19; cytokeratin 19; cytokeratin-19; keratin, type I, 40-kd; 40-kDa keratin intermediate filament
UniProt Protein Name:Keratin, type I cytoskeletal 19
UniProt Synonym Protein Names:Cytokeratin-19; CK-19; Keratin-19
Protein Family:Keratin
UniProt Gene Name:KRT19  
UniProt Entry Name:K1C19_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 19 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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