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Vimentin (Phospho-Ser83) Colorimetric Cell-Based ELISA Kit

SKU:
CBCAB01487
Product Type:
ELISA Kit
ELISA Type:
Cell Based Phospho Specific
Research Area:
Immunology
Reactivity:
Human
Mouse
Rat
Detection Method:
Colorimetric
$719
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Description

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Vimentin (Phospho-Ser83)Colorimetric Cell-Based ELISA Kit

The Vimentin Phospho-Ser83 Colorimetric Cell-Based ELISA Kit is a cutting-edge tool for researchers looking to analyze the phosphorylation status of vimentin at serine 83 in cell lysates. This kit provides high sensitivity and specificity, allowing for accurate and reproducible results in a variety of experimental settings.Vimentin is a key intermediate filament protein involved in maintaining cell structure, motility, and signaling. Phosphorylation of vimentin at serine 83 plays a crucial role in regulating its function and dynamics, making it a valuable target for studying cell processes such as migration, adhesion, and differentiation.

By using the Vimentin Phospho-Ser83 Colorimetric Cell-Based ELISA Kit, researchers can gain insight into the phosphorylation status of vimentin in response to various stimuli or treatments, providing valuable information for understanding cellular mechanisms and potential therapeutic targets.

Product Name:Vimentin (Phospho-Ser83) Colorimetric Cell-Based ELISA
Product Code:CBCAB01487
ELISA Type:Cell-Based
Target:Vimentin (Phospho-Ser83)
Reactivity:Human, Mouse, Rat
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nm
Format:2 x 96-Well Microplates

The Vimentin (Phospho-Ser83) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect Vimentin protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated Vimentin in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on Vimentin phosphorylation.

Qualitative determination of Vimentin (Phospho-Ser83) concentration is achieved by an indirect ELISA format. In essence, Vimentin (Phospho-Ser83) is captured by Vimentin (Phospho-Ser83)-specific primary (1ø) antibodies while the HRP-conjugated secondary (2ø) antibodies bind the Fc region of the 1ø antibody. Through this binding, the HRP enzyme conjugated to the 2ø 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: 7431, UniProt ID: P08670, OMIM: 193060, Unigene: Hs.642813
Gene Symbol:VIM
Sub Type:Phospho
UniProt Protein Function:Vimentin: an intermediate filament protein. Intermediate filament proteins are expressed in a tissue-specific manner. Desmin is the subunit specific for muscle and vimentin the subunit specific for mesenchymal tissue.
UniProt Protein Details:

Protein type:Cytoskeletal; Motility/polarity/chemotaxis

Chromosomal Location of Human Ortholog: 10p13

Cellular Component: intermediate filament cytoskeleton; neuron projection; focal adhesion; cytoskeleton; cytoplasm; leading edge; plasma membrane; intermediate filament; peroxisome; cytosol

Molecular Function:protein C-terminus binding; identical protein binding; protein binding; structural constituent of cytoskeleton; double-stranded RNA binding; glycoprotein binding; structural constituent of eye lens

Biological Process: Bergmann glial cell differentiation; viral reproduction; apoptosis; intermediate filament organization; cell motility; astrocyte development; cell structure disassembly during apoptosis; muscle filament sliding

Disease: Cataract 30

NCBI Summary:This gene encodes a type III intermediate filament protein. Intermediate filaments, along with microtubules and actin microfilaments, make up the cytoskeleton. The encoded protein is responsible for maintaining cell shape and integrity of the cytoplasm, and stabilizing cytoskeletal interactions. This protein is involved in neuritogenesis and cholesterol transport and functions as an organizer of a number of other critical proteins involved in cell attachment, migration, and signaling. Bacterial and viral pathogens have been shown to attach to this protein on the host cell surface. Mutations in this gene are associated with congenital cataracts in human patients. [provided by RefSeq, Aug 2017]
UniProt Code:P08670
NCBI GenInfo Identifier:55977767
NCBI Gene ID:7431
NCBI Accession:P08670.4
UniProt Related Accession:P08670
Molecular Weight:
NCBI Full Name:Vimentin
NCBI Synonym Full Names:vimentin
NCBI Official Symbol:VIM  
NCBI Protein Information:vimentin
UniProt Protein Name:Vimentin
Protein Family:Vimentin
UniProt Gene Name:VIM  
UniProt Entry Name:VIME_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-Vimentin (Phospho-Ser83) Antibody, Anti-Vimentin 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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