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TIMP4 Colorimetric Cell-Based ELISA Kit

SKU:
CBCAB00886
Product Type:
ELISA Kit
ELISA Type:
Cell Based
Reactivity:
Human
Mouse
Rat
Detection Method:
Colorimetric
€599
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Description

system_update_altDatasheetMSDS

TIMP4 Colorimetric Cell-Based ELISA Kit

The TIMP4 Colorimetric Cell-Based ELISA Kit is a cutting-edge tool for studying the tissue inhibitor of metalloproteinase 4 (TIMP-4) levels in cell culture samples. This kit offers exceptional sensitivity and specificity, allowing for precise and reliable detection of TIMP-4 in a variety of cell types.TIMP-4 is a key regulator of extracellular matrix remodeling and has been implicated in various diseases, including cancer, arthritis, and cardiovascular disorders.

By accurately measuring TIMP-4 levels, researchers can gain valuable insights into the mechanisms underlying these conditions and identify potential therapeutic targets.With its user-friendly protocol and high-performance capabilities, the TIMP4 Colorimetric Cell-Based ELISA Kit is the perfect choice for researchers looking to advance their understanding of TIMP-4 biology and its implications in disease pathology.

Product Name:TIMP4 Colorimetric Cell-Based ELISA
Product Code:CBCAB00886
ELISA Type:Cell-Based
Target:TIMP4
Reactivity:Human, Mouse, Rat
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:96-Well Microplate

The TIMP4 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect TIMP4 protein expression profile in cells. The kit can be used for measuring the relative amounts of TIMP4 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on TIMP4.

Qualitative determination of TIMP4 concentration is achieved by an indirect ELISA format. In essence, TIMP4 is captured by TIMP4-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: 7079, UniProt ID: Q99727, OMIM: 601915, Unigene: Hs.591665
Gene Symbol:TIMP4
Sub Type:None
UniProt Protein Function:TIMP4: Complexes with metalloproteinases (such as collagenases) and irreversibly inactivates them by binding to their catalytic zinc cofactor. Known to act on MMP-1, MMP-2, MMP-3, MMP-7 and MMP- 9. Belongs to the protease inhibitor I35 (TIMP) family.
UniProt Protein Details:

Protein type:Secreted; Secreted, signal peptide

Chromosomal Location of Human Ortholog: 3p25

Cellular Component: extracellular space; proteinaceous extracellular matrix; sarcomere

Molecular Function:metal ion binding; metalloendopeptidase inhibitor activity; protease binding

Biological Process: central nervous system development; negative regulation of membrane protein ectodomain proteolysis; negative regulation of metalloenzyme activity; Notch signaling pathway; ovulation cycle; response to cytokine stimulus; response to drug; response to lipopolysaccharide; response to organic substance; response to peptide hormone stimulus

NCBI Summary:This gene belongs to the TIMP gene family. The proteins encoded by this gene family are inhibitors of the matrix metalloproteinases, a group of peptidases involved in degradation of the extracellular matrix. The secreted, netrin domain-containing protein encoded by this gene is involved in regulation of platelet aggregation and recruitment and may play role in hormonal regulation and endometrial tissue remodeling. [provided by RefSeq, Jul 2008]
UniProt Code:Q99727
NCBI GenInfo Identifier:3915135
NCBI Gene ID:7079
NCBI Accession:Q99727.1
UniProt Secondary Accession:Q99727,B2R7K6,
UniProt Related Accession:Q99727
Molecular Weight:25,503 Da
NCBI Full Name:Metalloproteinase inhibitor 4
NCBI Synonym Full Names:TIMP metallopeptidase inhibitor 4
NCBI Official Symbol:TIMP4  
NCBI Protein Information:metalloproteinase inhibitor 4
UniProt Protein Name:Metalloproteinase inhibitor 4
UniProt Synonym Protein Names:Tissue inhibitor of metalloproteinases 4; TIMP-4
Protein Family:Metalloproteinase inhibitor
UniProt Gene Name:TIMP4  
UniProt Entry Name:TIMP4_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-TIMP4 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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