Actin-gamma2 Colorimetric Cell-Based ELISA Kit

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SKU:
CBCAB00513
Product Type:
ELISA Kit
ELISA Type:
Cell Based
Reactivity:
Human
Reactivity:
Mouse
Reactivity:
Rat
Detection Method:
Colorimetric

Description

system_update_altDatasheet
Product Name:Actin-gamma2 Colorimetric Cell-Based ELISA
Product Code:CBCAB00513
ELISA Type:Cell-Based
Target:Actin-gamma2
Reactivity:Human, Mouse, Rat
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:96-Well Microplate

The Actin-gamma2 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect Actin-gamma2 protein expression profile in cells. The kit can be used for measuring the relative amounts of Actin-gamma2 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on Actin-gamma2.

Qualitative determination of Actin-gamma2 concentration is achieved by an indirect ELISA format. In essence, Actin-gamma2 is captured by Actin-gamma2-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: 72, UniProt ID: P63267, OMIM: 102545, Unigene: Hs.516105
Gene Symbol:ACTG2
Sub Type:None
UniProt Protein Function:ACTG2: Actins are highly conserved proteins that are involved in various types of cell motility and are ubiquitously expressed in all eukaryotic cells. Polymerization of globular actin (G-actin) leads to a structural filament (F-actin) in the form of a two-stranded helix. Each actin can bind to 4 others. Belongs to the actin family.
UniProt Protein Details:

Protein type:Contractile; Cytoskeletal; Motility/polarity/chemotaxis

Chromosomal Location of Human Ortholog: 2p13.1

Cellular Component: cytoplasm; cytosol; extracellular space; filopodium; lamellipodium

Biological Process: muscle contraction

Disease: Visceral Myopathy

NCBI Summary:Actins are highly conserved proteins that are involved in various types of cell motility and in the maintenance of the cytoskeleton. Three types of actins, alpha, beta and gamma, have been identified in vertebrates. Alpha actins are found in muscle tissues and are a major constituent of the contractile apparatus. The beta and gamma actins co-exist in most cell types as components of the cytoskeleton and as mediators of internal cell motility. This gene encodes actin gamma 2; a smooth muscle actin found in enteric tissues. Alternative splicing results in multiple transcript variants encoding distinct isoforms. Based on similarity to peptide cleavage of related actins, the mature protein of this gene is formed by removal of two N-terminal peptides.[provided by RefSeq, Dec 2010]
UniProt Code:P63267
NCBI GenInfo Identifier:54036679
NCBI Gene ID:72
NCBI Accession:P63267.1
UniProt Secondary Accession:P63267,P12718, Q504R1, Q6FI22, B2R7E7, B4E315, D6W5H8 E9PG30,
UniProt Related Accession:P63267
Molecular Weight:37,083 Da
NCBI Full Name:Actin, gamma-enteric smooth muscle
NCBI Synonym Full Names:actin, gamma 2, smooth muscle, enteric
NCBI Official Symbol:ACTG2  
NCBI Official Synonym Symbols:ACT; ACTE; VSCM; ACTA3; ACTL3; ACTSG  
NCBI Protein Information:actin, gamma-enteric smooth muscle
UniProt Protein Name:Actin, gamma-enteric smooth muscle
UniProt Synonym Protein Names:Alpha-actin-3; Gamma-2-actin; Smooth muscle gamma-actin
Protein Family:Actin
UniProt Gene Name:ACTG2  
UniProt Entry Name:ACTH_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-Actin-gamma2 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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