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MMP-8 Colorimetric Cell-Based ELISA Kit

SKU:
CBCAB00767
Product Type:
ELISA Kit
ELISA Type:
Cell Based
Research Area:
Cell Biology
Reactivity:
Human
Mouse
Rat
Detection Method:
Colorimetric
$719
Frequently bought together:

Description

system_update_altDatasheetMSDS

MMP-8 Colorimetric Cell-Based ELISA Kit

The MMP-8 Colorimetric Cell-Based ELISA Kit is a powerful tool for researchers looking to accurately detect MMP-8 levels in cell culture supernatants. This kit offers high sensitivity and specificity, ensuring precise and reliable results for a variety of research applications.MMP-8, also known as Collagenase-2, is a key enzyme involved in the breakdown of extracellular matrix proteins, making it crucial in processes such as tissue remodeling and wound healing. Dysregulation of MMP-8 activity has been linked to various diseases, including arthritis, cancer, and cardiovascular disorders, highlighting its importance as a potential biomarker for disease progression and therapeutic development.

With the MMP-8 Colorimetric Cell-Based ELISA Kit, researchers can easily and accurately measure MMP-8 levels in cell cultures, allowing for a better understanding of its role in disease pathogenesis and the development of targeted treatment strategies. Trust in this kit for robust and reproducible results in your research endeavors.

Product Name:MMP-8 Colorimetric Cell-Based ELISA
Product Code:CBCAB00767
ELISA Type:Cell-Based
Target:MMP-8
Reactivity:Human, Mouse, Rat
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:96-Well Microplate

The MMP-8 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect MMP-8 protein expression profile in cells. The kit can be used for measuring the relative amounts of MMP-8 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on MMP-8.

Qualitative determination of MMP-8 concentration is achieved by an indirect ELISA format. In essence, MMP-8 is captured by MMP-8-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: 4317, UniProt ID: P22894, OMIM: 120355, Unigene: Hs.161839
Gene Symbol:MMP8
Sub Type:None
UniProt Protein Function:MMP8: Can degrade fibrillar type I, II, and III collagens. Belongs to the peptidase M10A family.
UniProt Protein Details:

Protein type:Protease; EC 3.4.24.34; Secreted, signal peptide; Secreted

Chromosomal Location of Human Ortholog: 11q22.3

Cellular Component: extracellular space; proteinaceous extracellular matrix; extracellular region

Molecular Function:zinc ion binding; serine-type endopeptidase activity; metalloendopeptidase activity; calcium ion binding

Biological Process: extracellular matrix disassembly; collagen catabolic process; extracellular matrix organization and biogenesis; proteolysis

NCBI Summary:Proteins of the matrix metalloproteinase (MMP) family are involved in the breakdown of extracellular matrix in normal physiological processes, such as embryonic development, reproduction, and tissue remodeling, as well as in disease processes, such as arthritis and metastasis. Most MMP's are secreted as inactive proproteins which are activated when cleaved by extracellular proteinases. However, the enzyme encoded by this gene is stored in secondary granules within neutrophils and is activated by autolytic cleavage. Its function is degradation of type I, II and III collagens. The gene is part of a cluster of MMP genes which localize to chromosome 11q22.3. [provided by RefSeq, Jul 2008]
UniProt Code:P22894
NCBI GenInfo Identifier:116862
NCBI Gene ID:4317
NCBI Accession:P22894.1
UniProt Secondary Accession:P22894,Q45F99,
UniProt Related Accession:P22894
Molecular Weight:53,412 Da
NCBI Full Name:Neutrophil collagenase
NCBI Synonym Full Names:matrix metallopeptidase 8 (neutrophil collagenase)
NCBI Official Symbol:MMP8  
NCBI Official Synonym Symbols:HNC; CLG1; MMP-8; PMNL-CL  
NCBI Protein Information:neutrophil collagenase; PMNL collagenase; matrix metalloproteinase-8; matrix metalloproteinase 8 (neutrophil collagenase)
UniProt Protein Name:Neutrophil collagenase
UniProt Synonym Protein Names:Matrix metalloproteinase-8; MMP-8; PMNL collagenase; PMNL-CL
Protein Family:Neutrophil collagenase
UniProt Gene Name:MMP8  
UniProt Entry Name:MMP8_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-MMP-8 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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