Figure 1. Treatment of HeLa cells with IFN-α stimulates phosphorylation of Stat3 at Tyr705 as detected by PathScan® Phospho-Stat3 (Tyr705) Sandwich ELISA Kit #7300, but does not affect the level of β-actin as detected by PathScan® Total β-Actin Sandwich ELISA Kit #7880. HeLa cells were treated with 100 ng/ml IFN-α for ten minutes at 37ºC before lysis. Absorbance at 450 nm is shown in the top figure, while the corresponding western blots using Phospho-Stat3 (Tyr705) (3E2) Mouse mAb #9138 (right panel) or Total β-Actin (13E5) Rabbit mAb #4970 (left panel) are shown in the bottom figure.
Figure 2. The relationship between the protein concentration of the lysate from HeLa cells and the absorbance at 450 nm is shown.
|REACTIVITY||H M R Hm Mk|
|Product Includes||Volume||Solution Color|
|β-Actin Rabbit mAb Coated Microwells||96 tests|
|Pan-Actin Mouse Detection mAb||1 ea||Green (Lyophilized)|
|Anti-mouse IgG, HRP-linked Antibody (ELISA Formulated)||1 ea||Red (Lyophilized)|
|Detection Antibody Diluent||11 ml||Green|
|HRP Diluent||11 ml||Red|
|TMB Substrate 7004||11 ml|
|STOP Solution 7002||11 ml|
|Sealing Tape||2 ea|
|ELISA Wash Buffer (20X) 9801||25 ml|
|ELISA Sample Diluent||25 ml||Blue|
|Cell Lysis Buffer (10X) 9803||15 ml|
The PathScan® Total β-Actin Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of β-actin. A β-actin rabbit antibody has been coated onto the microwells. After incubation with cell lysates, β-actin is captured by the coated antibody. Following extensive washing, a pan-actin mouse detection antibody is added to detect the captured β-actin. An anti-mouse IgG, HRP-linked antibody is then used to recognize the bound detection antibody. HRP substrate (TMB) is added to develop color. The magnitude of the absorbance for this developed color is proportional to the quantity of β-actin.
Antibodies in kit are custom formulations specific to kit.
NOTE: Prepare solutions with purified water.
*NOTE: Some PathScan® ELISA Kits may include HRP-Linked Streptavidin in place of HRP-Linked Antibody.
NOTE: Initial color of positive reaction is blue, which changes to yellow upon addition of STOP Solution.
posted November 2013
Protocol Id: 204
CST's PathScan® Total β-Actin Sandwich ELISA Kit detects endogenous levels of β-actin. As shown in Figure 1, β-actin is readily detected in HeLa cells using the PathScan® Total β-Actin Sandwich ELISA Kit. Total levels of β-actin remain unchanged after IFN-α treatment as shown by western analysis. The PathScan® Total β-Actin Sandwich ELISA Kit does not cross-react with α-smooth muscle actin, α-sarcomeric muscle actin or γ-actin. This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.
Human, Mouse, Rat, Hamster, Monkey
Actin, a ubiquitous eukaryotic protein, is the major component of the cytoskeleton. At least six isoforms are known in mammals. Nonmuscle β- and γ-actin, also known as cytoplasmic actin, are ubiquitously expressed, controlling cell structure and motility (1). While all actin isoforms are highly homologous, cytoplasmic β- and γ-actin protein sequences differ by only four biochemically similar amino acids (2). For this reason, antibodies raised to β-actin may cross-react with γ-actin, and vice versa. α-cardiac and α-skeletal actin are expressed in striated cardiac and skeletal muscles, respectively; two smooth muscle actins, α- and γ-actin, are found primarily in vascular smooth muscle and enteric smooth muscle, respectively. These actin isoforms regulate the contractile potential of muscle cells (1). Actin exists mainly as a fibrous polymer, F-actin. In response to cytoskeletal reorganizing signals during processes such as cytokinesis, endocytosis, or stress, cofilin promotes fragmentation and depolymerization of F-actin, resulting in an increase in the monomeric globular form, G-actin (3). The ARP2/3 complex stabilizes F-actin fragments and promotes formation of new actin filaments (3). Research studies have shown that actin is hyperphosphorylated in primary breast tumors (4). Cleavage of actin under apoptotic conditions has been observed in vitro and in cardiac and skeletal muscle, as shown in research studies (5-7). Actin cleavage by caspase-3 may accelerate ubiquitin/proteasome-dependent muscle proteolysis (7).
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