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Stefin A Colorimetric Cell-Based ELISA Kit

SKU:
CBCAB00872
Product Type:
ELISA Kit
ELISA Type:
Cell Based
Research Area:
Cell Biology
Reactivity:
Human
Detection Method:
Colorimetric
€599
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Description

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Stefin A Colorimetric Cell-Based ELISA Kit

The Stefin A Colorimetric Cell-Based ELISA Kit is a cutting-edge assay designed for the accurate detection of Stefin A levels in cell lysates and tissue homogenates. This kit offers exceptional sensitivity and specificity, providing researchers with reliable and reproducible results for their studies.Stefin A, also known as cystatin A, is a key protein involved in the regulation of protease activity and plays a critical role in processes such as cell growth, differentiation, and apoptosis. Dysregulation of Stefin A has been linked to various diseases, including cancer, neurodegenerative disorders, and inflammatory conditions, making it a valuable biomarker for research and potential therapeutic development.

With its easy-to-use protocol and robust performance, the Stefin A Colorimetric Cell-Based ELISA Kit is an essential tool for investigating the role of Stefin A in disease pathology and exploring novel treatment strategies. Order yours today and take your research to the next level.

Product Name:Stefin A Colorimetric Cell-Based ELISA
Product Code:CBCAB00872
ELISA Type:Cell-Based
Target:Stefin A
Reactivity:Human
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:96-Well Microplate

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

Qualitative determination of Stefin A concentration is achieved by an indirect ELISA format. In essence, Stefin A is captured by Stefin A-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: 1475, UniProt ID: P01040, OMIM: 184600, Unigene: Hs.518198
Gene Symbol:CSTA
Sub Type:None
UniProt Protein Function:CSTA: This is an intracellular thiol proteinase inhibitor. Has an important role in desmosome-mediated cell-cell adhesion in the lower levels of the epidermis. Defects in CSTA are the cause of ichthyosis exfoliative autosomal recessive ichthyosis bullosa of Siemens-like (AREI). A form of congenital exfoliative ichthyosis, sharing some features with ichthyosis bullosa of Siemens and annular epidermolytic ichthyosis. AREI presents shortly after birth as dry, scaly skin over most of the body with coarse peeling of non- erythematous skin on the palms and soles, which is exacerbated by excessive moisture and minor trauma. Electron microscopy analysis of skin biopsies, reveals mostly normal-appearing upper layers of the epidermis, but prominent intercellular edema of the basal and suprabasal cell layers with aggregates of tonofilaments in the basal keratinocytes. Belongs to the cystatin family.
UniProt Protein Details:

Chromosomal Location of Human Ortholog: 3q21

Cellular Component: cornified envelope; cytoplasm; extracellular space; nucleus

Molecular Function:cysteine protease inhibitor activity; protease binding; protein binding, bridging; structural molecule activity

Biological Process: cell-cell adhesion; keratinocyte differentiation; negative regulation of peptidase activity; negative regulation of proteolysis; peptide cross-linking

Disease: Exfoliative Ichthyosis, Autosomal Recessive, Ichthyosis Bullosa Of Siemens-like

NCBI Summary:The cystatin superfamily encompasses proteins that contain multiple cystatin-like sequences. Some of the members are active cysteine protease inhibitors, while others have lost or perhaps never acquired this inhibitory activity. There are three inhibitory families in the superfamily, including the type 1 cystatins (stefins), type 2 cystatins, and kininogens. This gene encodes a stefin that functions as a cysteine protease inhibitor, forming tight complexes with papain and the cathepsins B, H, and L. The protein is one of the precursor proteins of cornified cell envelope in keratinocytes and plays a role in epidermal development and maintenance. Stefins have been proposed as prognostic and diagnostic tools for cancer. [provided by RefSeq, Jul 2008]
UniProt Code:P01040
NCBI GenInfo Identifier:118177
NCBI Gene ID:1475
NCBI Accession:P01040.1
UniProt Secondary Accession:P01040,Q6IB90,
UniProt Related Accession:P01040
Molecular Weight:11,006 Da
NCBI Full Name:Cystatin-A
NCBI Synonym Full Names:cystatin A
NCBI Official Symbol:CSTA  
NCBI Official Synonym Symbols:AREI; PSS4; STF1; STFA  
NCBI Protein Information:cystatin-A
UniProt Protein Name:Cystatin-A
UniProt Synonym Protein Names:Cystatin-AS; Stefin-A
Protein Family:Cystatin
UniProt Gene Name:CSTA  
UniProt Entry Name:CYTA_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-Stefin A 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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