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Actinin alpha-2/3 Colorimetric Cell-Based ELISA

SKU:
CBCAB01005
Product Type:
ELISA Kit
ELISA Type:
Cell Based
Reactivity:
Human
Mouse
Rat
Detection Method:
Colorimetric
€599
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Description

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Actinin alpha-2/3 Colorimetric Cell-Based ELISA

The ACTN2/3 (Actinin Alpha 2/3) Colorimetric Cell-Based ELISA Kit is specifically designed for the accurate detection of ACTN2/3 levels in cell lysates, cell culture supernatants, and tissue homogenates. This kit offers high sensitivity and specificity, ensuring precise and reliable results for a wide range of research applications.Actinin Alpha 2 and 3 are critical proteins involved in cell adhesion and cytoskeletal organization, playing key roles in cell migration, differentiation, and signaling. Dysregulation of ACTN2/3 expression has been linked to various diseases, including muscular dystrophy, heart disease, and cancer, making this kit an invaluable tool for studying the molecular mechanisms underlying these conditions and identifying potential therapeutic targets.

With easy-to-follow protocols and quick assay turnaround times, the ACTN2/3 Colorimetric Cell-Based ELISA Kit provides researchers with a convenient and efficient method for studying the expression levels of these important actin-binding proteins in biological samples.

Product Name:Actinin alpha-2/3 Colorimetric Cell-Based ELISA
Product Code:CBCAB01005
ELISA Type:Cell-Based
Target:Actinin alpha-2/3
Reactivity:Human, Mouse, Rat
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:96-Well Microplate

The Actinin alpha-2/3 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect Actinin alpha-2/3 protein expression profile in cells. The kit can be used for measuring the relative amounts of Actinin alpha-2/3 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on Actinin alpha-2/3.

Qualitative determination of Actinin alpha-2/3 concentration is achieved by an indirect ELISA format. In essence, Actinin alpha-2/3 is captured by Actinin alpha-2/3-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: 88/89, UniProt ID: P35609/Q08043, OMIM: 102573/612158/102574, Unigene: Hs.498178/Hs.654432
Gene Symbol:ACTN2
Sub Type:None
UniProt Protein Function:ACTN2: F-actin cross-linking protein which is thought to anchor actin to a variety of intracellular structures. This is a bundling protein. Defects in ACTN2 are the cause of cardiomyopathy dilated type 1AA (CMD1AA). Dilated cardiomyopathy is a disorder characterized by ventricular dilation and impaired systolic function, resulting in congestive heart failure and arrhythmia. Patients are at risk of premature death. Belongs to the alpha-actinin family.
UniProt Protein Details:

Protein type:Motility/polarity/chemotaxis; Cytoskeletal

Chromosomal Location of Human Ortholog: 1q42-q43

Cellular Component: cortical actin cytoskeleton; cytoskeleton; focal adhesion; extracellular region; dendritic spine; actin filament; pseudopodium; Z disc; cytosol; filopodium

Molecular Function:actin filament binding; protein dimerization activity; integrin binding; identical protein binding; protein binding; LIM domain binding; structural constituent of muscle; cytoskeletal protein binding; titin binding; calcium ion binding; FATZ binding; thyroid hormone receptor coactivator activity

Biological Process: focal adhesion formation; platelet activation; negative regulation of potassium ion transport; positive regulation of potassium ion transport; muscle filament sliding; protein homotetramerization; regulation of apoptosis; synaptic transmission; regulation of membrane potential; platelet degranulation; microspike biogenesis; blood coagulation; cell adhesion

Disease: Cardiomyopathy, Dilated, 1aa

NCBI Summary:Alpha actinins belong to the spectrin gene superfamily which represents a diverse group of cytoskeletal proteins, including the alpha and beta spectrins and dystrophins. Alpha actinin is an actin-binding protein with multiple roles in different cell types. In nonmuscle cells, the cytoskeletal isoform is found along microfilament bundles and adherens-type junctions, where it is involved in binding actin to the membrane. In contrast, skeletal, cardiac, and smooth muscle isoforms are localized to the Z-disc and analogous dense bodies, where they help anchor the myofibrillar actin filaments. This gene encodes a muscle-specific, alpha actinin isoform that is expressed in both skeletal and cardiac muscles. Several transcript variants encoding different isoforms have been found for this gene. [provided by RefSeq, May 2013]
UniProt Code:P35609
NCBI GenInfo Identifier:543742
NCBI Gene ID:88
NCBI Accession:P35609.1
UniProt Secondary Accession:P35609,Q86TF4, Q86TI8, B1ANE4, B2RCS5,
UniProt Related Accession:P35609
Molecular Weight:103,920 Da
NCBI Full Name:Alpha-actinin-2
NCBI Synonym Full Names:actinin, alpha 2
NCBI Official Symbol:ACTN2  
NCBI Official Synonym Symbols:CMD1AA  
NCBI Protein Information:alpha-actinin-2; F-actin cross-linking protein; alpha-actinin skeletal muscle
UniProt Protein Name:Alpha-actinin-2
UniProt Synonym Protein Names:Alpha-actinin skeletal muscle isoform 2; F-actin cross-linking protein
Protein Family:Alpha-actinin
UniProt Gene Name:ACTN2  
UniProt Entry Name:ACTN2_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-Actinin alpha-2/3 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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