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Caldesmon Colorimetric Cell-Based ELISA Kit

SKU:
CBCAB00555
Product Type:
ELISA Kit
ELISA Type:
Cell Based
Reactivity:
Human
Mouse
Rat
Detection Method:
Colorimetric
€599
Frequently bought together:

Description

system_update_altDatasheetMSDS

Caldesmon Colorimetric Cell-Based ELISA Kit

The Caldesmon Colorimetric Cell-Based ELISA Kit from Assay Genie is a powerful tool for the detection of caldesmon levels in cell cultures. This kit offers high sensitivity and specificity, allowing for accurate and reproducible results in various research applications.Caldesmon is a key regulatory protein in smooth muscle cells, playing a crucial role in regulating cell motility and contraction. Dysregulation of caldesmon has been linked to conditions such as cancer, cardiovascular diseases, and muscular disorders, making it an important biomarker for studying disease mechanisms and therapeutic development.

With the Assay Genie Caldesmon Colorimetric Cell-Based ELISA Kit, researchers can confidently measure caldesmon levels in cell cultures with precision and efficiency, advancing their understanding of cellular processes and disease pathways.

Product Name:Caldesmon Colorimetric Cell-Based ELISA
Product Code:CBCAB00555
ELISA Type:Cell-Based
Target:Caldesmon
Reactivity:Human, Mouse, Rat
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:96-Well Microplate

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

Qualitative determination of Caldesmon concentration is achieved by an indirect ELISA format. In essence, Caldesmon is captured by Caldesmon-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: 800, UniProt ID: Q05682, OMIM: 114213, Unigene: Hs.490203
Gene Symbol:CALD1
Sub Type:None
UniProt Protein Function:Caldesmon: a cytoskeletal protein implicated in the regulation of actomyosin interactions in smooth muscle and nonmuscle cells. May act as a bridge between myosin and actin filaments. Stimulates actin binding of tropomyosin which increases the stabilization of actin filament structure. In muscle tissues, inhibits the actomyosin ATPase by binding to F-actin. This inhibition is attenuated by calcium-calmodulin and is potentiated by tropomyosin. Interacts with actin, myosin, two molecules of tropomyosin and with calmodulin. Also plays an essential role during cellular mitosis and receptor capping. Five alternatively spliced isoforms have been described.
UniProt Protein Details:

Protein type:Actin-binding

Chromosomal Location of Human Ortholog: 7q33

Cellular Component: actin cap; actin cytoskeleton; cytoskeleton; cytosol; myofibril; plasma membrane

Molecular Function:actin binding; calmodulin binding; myosin binding; protein binding; tropomyosin binding

Biological Process: cell motility; muscle contraction

NCBI Summary:This gene encodes a calmodulin- and actin-binding protein that plays an essential role in the regulation of smooth muscle and nonmuscle contraction. The conserved domain of this protein possesses the binding activities to Ca(2+)-calmodulin, actin, tropomyosin, myosin, and phospholipids. This protein is a potent inhibitor of the actin-tropomyosin activated myosin MgATPase, and serves as a mediating factor for Ca(2+)-dependent inhibition of smooth muscle contraction. Alternative splicing of this gene results in multiple transcript variants encoding distinct isoforms. [provided by RefSeq, Jul 2008]
UniProt Code:Q05682
NCBI GenInfo Identifier:338817891
NCBI Gene ID:800
NCBI Accession:Q05682.3
UniProt Secondary Accession:Q05682,Q13978, Q13979, Q14741, Q14742, Q9UD91, A8K0X1
UniProt Related Accession:Q05682
Molecular Weight:65,620 Da
NCBI Full Name:Caldesmon
NCBI Synonym Full Names:caldesmon 1
NCBI Official Symbol:CALD1  
NCBI Official Synonym Symbols:CDM; HCAD; LCAD; H-CAD; L-CAD; NAG22  
NCBI Protein Information:caldesmon
UniProt Protein Name:Caldesmon
Protein Family:Caldesmon
UniProt Gene Name:CALD1  
UniProt Entry Name:CALD1_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-Caldesmon 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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