Cell Signaling Technology

Product Pathways - PathScan ELISA

PathScan® Total β-Actin Sandwich ELISA Kit #7880

When ordering five or more kits, please contact us for processing time and pricing at sales@cellsignal.com.

Kit Includes Volume Solution Color
Beta-Actin Ab Coated Microwells 96 tests
Pan-Actin Detection Ab 11 ml Green
Anti-mouse IgG, HRP-linked Antibody 11 ml Red
TMB Substrate #7004 11 ml Colorless
STOP Solution #7002 11 ml Colorless
Sealing Tape 2 sheets
ELISA Wash Buffer (20X) 25 ml Colorless
ELISA Sample Diluent 25 ml Blue
Cell Lysis Buffer (10X) #9803 15 ml Yellowish

Note: 12 8-well modules –Each module is designed to break apart for 8 tests.
Note: Kit should be stored at 4°C with the exception of Cell Lysis Buffer, which is stored at –20°C (packaged separately).

Species Cross-Reactivity

H M R Hm Mk

Reactivity Key:  H=Human  M=Mouse  R=Rat  Hm=Hamster  Mk=Monkey
Species enclosed in parentheses are predicted to react based on 100% sequence homology.

Description

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.

Specificity / Sensitivity

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.

ELISA - Western correlation

ELISA - Western correlation

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.

Sensitivity

Sensitivity

Figure 2. The relationship between the protein concentration of the lysate from HeLa cells and the absorbance at 450 nm is shown.

Background

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 predominantly expressed in nonmuscle cells, controlling cell structure and motility (1). 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 (2). The ARP2/3 complex stabilizes F-actin fragments and promotes formation of new actin filaments (2). Research studies have shown that actin is hyperphosphorylated in primary breast tumors (3). Cleavage of actin under apoptotic conditions has been observed in vitro and in cardiac and skeletal muscle, as shown in research studies (4-6). Actin cleavage by caspase-3 may accelerate ubiquitin/proteasome-dependent muscle proteolysis (6).

  1. Herman, I.M. (1993) Curr. Opin. Cell Biol. 5, 48-55.
  2. Condeelis, J. (2001) Trends Cell Biol. 11, 288-293.
  3. Lim, Y.P. et al. (2004) Clin. Cancer Res. 10, 3980-3987.
  4. Kayalar, C. et al. (1996) Proc. Natl. Acad. Sci. USA. 93, 2234-2238.
  5. Communal, C. et al. (2002) Proc. Natl. Acad. Sci. USA. 99, 6252-6256.
  6. Du, J. et al. (2004) J. Clin. Invest. 113, 115-123.

Application References

Have you published research involving the use of our products? If so we'd love to hear about it. Please let us know!

Protocols

Companion Products

For Research Use Only. Not For Use In Diagnostic Procedures.

Products