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Calnexin Colorimetric Cell-Based ELISA Kit

SKU:
CBCAB00556
Product Type:
ELISA Kit
ELISA Type:
Cell Based
Research Area:
Immunology
Reactivity:
Human
Mouse
Rat
Detection Method:
Colorimetric
€599
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Description

system_update_altDatasheet

Calnexin Colorimetric Cell-Based ELISA Kit

The Calnexin Colorimetric Cell-Based ELISA Kit from Assay Genie is a powerful tool for researchers looking to accurately detect and quantify calnexin levels in cell samples. This kit offers high sensitivity and specificity, ensuring reliable and reproducible results for a variety of research applications.Calnexin is a critical protein involved in protein folding and quality control within the endoplasmic reticulum. Dysregulation of calnexin has been implicated in a range of diseases, including cancer, neurodegenerative disorders, and inflammatory conditions.

By using the Calnexin Colorimetric Cell-Based ELISA Kit, researchers can better understand the role of calnexin in these diseases and potentially identify new therapeutic targets.Overall, the Calnexin Colorimetric Cell-Based ELISA Kit provides researchers with a valuable tool to study the function and regulation of calnexin in various cellular processes and disease states. Its high sensitivity and specificity make it an essential addition to any lab conducting research in cell biology or protein quality control mechanisms.

Product Name:Calnexin Colorimetric Cell-Based ELISA
Product Code:CBCAB00556
ELISA Type:Cell-Based
Target:Calnexin
Reactivity:Human, Mouse, Rat
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:96-Well Microplate

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

Qualitative determination of Calnexin concentration is achieved by an indirect ELISA format. In essence, Calnexin is captured by Calnexin-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: 821, UniProt ID: P27824, OMIM: 114217, Unigene: Hs.567968
Gene Symbol:CANX
Sub Type:None
UniProt Protein Function:Calnexin: a calcium-binding protein of the calreticulin family. A type I membrane protein of the endoplasmic reticulum.Interacts with newly synthesized glycoproteins in the endoplasmic reticulum. May act in assisting protein assembly and/or in the retention within the ER of unassembled protein subunits. It seems to play a major role in the quality control apparatus of the ER by the retention of incorrectly folded proteins.
UniProt Protein Details:

Protein type:Endoplasmic reticulum; Calcium-binding; Motility/polarity/chemotaxis; Membrane protein, integral

Chromosomal Location of Human Ortholog: 5q35

Cellular Component: dendrite cytoplasm; endoplasmic reticulum membrane; protein complex; smooth endoplasmic reticulum; rough endoplasmic reticulum; endoplasmic reticulum lumen; endoplasmic reticulum; dendritic spine; cell soma; membrane; axon; melanosome; ribosome

Molecular Function:ionotropic glutamate receptor binding; protein binding; unfolded protein binding; apolipoprotein binding; calcium ion binding; carbohydrate binding; glycoprotein binding

Biological Process: antigen processing and presentation of peptide antigen via MHC class I; cellular protein metabolic process; protein folding; synaptic vesicle endocytosis; protein secretion; antigen processing and presentation of exogenous peptide antigen via MHC class II; protein amino acid N-linked glycosylation via asparagine; post-translational protein modification; aging

NCBI Summary:This gene encodes a member of the calnexin family of molecular chaperones. The encoded protein is a calcium-binding, endoplasmic reticulum (ER)-associated protein that interacts transiently with newly synthesized N-linked glycoproteins, facilitating protein folding and assembly. It may also play a central role in the quality control of protein folding by retaining incorrectly folded protein subunits within the ER for degradation. Alternatively spliced transcript variants encoding the same protein have been described. [provided by RefSeq, Jul 2008]
UniProt Code:P27824
NCBI GenInfo Identifier:543920
NCBI Gene ID:821
NCBI Accession:P27824.2
UniProt Secondary Accession:P27824,B2R5V8, B4DGP8, B4E2T8, D3DWQ3, D6R9K3,
UniProt Related Accession:P27824
Molecular Weight:55,598 Da
NCBI Full Name:Calnexin
NCBI Synonym Full Names:calnexin
NCBI Official Symbol:CANX  
NCBI Official Synonym Symbols:CNX; P90; IP90  
NCBI Protein Information:calnexin; major histocompatibility complex class I antigen-binding protein p88
UniProt Protein Name:Calnexin
UniProt Synonym Protein Names:IP90; Major histocompatibility complex class I antigen-binding protein p88; p90
UniProt Gene Name:CANX  
UniProt Entry Name:CALX_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-Calnexin 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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