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7881
PathScan® Total β-Actin Sandwich ELISA Antibody Pair
ELISA Kits

PathScan® Total β-Actin Sandwich ELISA Antibody Pair #7881

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Figure 1. The relationship between the protein concentration of the lysate from HeLa cells and the absorbance at 450 nm using the PathScan® Total β-Actin Sandwich ELISA Antibody Pair is shown.

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ELISA Antibody Pair

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

  1. 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix.
  2. Wash Buffer: 1X PBS/0.05% Tween® 20, (20X PBST #9809).
  3. Blocking Buffer: 1X PBS/0.05% Tween® 20, 1% BSA.

  4. 1X Cell Lysis Buffer: 10X Cell Lysis Buffer (#9803): To prepare 10 ml of 1X Cell Lysis Buffer, add 1 ml of 10X Cell Lysis Buffer to 9 ml of dH2O, mix. Buffer can be stored at 4°C for short-term use (1–2 weeks).

    Recommended: Add 1 mM phenylmethylsulfonyl fluoride (PMSF) (#8553) immediately before use.

  5. Bovine Serum Albumin (BSA): (#9998).
  6. TMB Substrate: (#7004).

  7. STOP Solution: (#7002)

    NOTE: Reagents should be made fresh daily.

B. Preparing Cell Lysates

For adherent cells

  1. Aspirate media when the culture reaches 80–90% confluence. Treat cells by adding fresh media containing regulator for desired time.
  2. Remove media and rinse cells once with ice-cold 1X PBS.
  3. Remove PBS and add 0.5 ml ice-cold 1X Cell Lysis Buffer plus 1 mM PMSF to each plate (10 cm diameter) and incubate the plate on ice for 5 min.
  4. Scrape cells off the plate and transfer to an appropriate tube. Keep on ice.
  5. Sonicate lysates on ice.
  6. Microcentrifuge for 10 min (x14,000 rpm) at 4°C and transfer the supernatant to a new tube. The supernatant is the cell lysate. Store at -80°C in single-use aliquots.

For suspension cells

  1. Remove media by low speed centrifugation (~1,200 rpm) when the culture reaches 0.5–1.0 x 106 viable cells/ml. Treat cells by adding fresh media containing regulator for desired time.
  2. Collect cells by low speed centrifugation (~1,200 rpm) and wash once with 5–10 ml ice-cold 1X PBS.
  3. Cells harvested from 50 ml of growth media can be lysed in 2.0 ml of 1X cell lysis buffer plus 1 mM PMSF.
  4. Sonicate lysates on ice.
  5. Microcentrifuge for 10 min (x14,000 rpm) at 4°C and transfer the supernatant to a new tube. The supernatant is the cell lysate. Store at -80°C in single-use aliquots.

C. Coating Procedure

  1. Rinse microplate with 200 µl of dH2O, discard liquid. Blot on paper towel to make sure wells are dry.
  2. Dilute capture antibody 1:100 in 1X PBS. For a single 96 well plate, add 100 µl of capture antibody stock to 9.9 ml 1X PBS. Mix well and add 100 µl/well. Cover plate and incubate overnight at 4°C (17–20 hr).
  3. After overnight coating, gently uncover plate and wash wells:
    1. Discard plate contents into a receptacle.
    2. Wash four times with wash buffer, 200 µl each time per well. For each wash, strike plates on fresh paper towels hard enough to remove the residual solution in each well, but do not allow wells to completely dry at any time.
    3. Clean the underside of all wells with a lint-free tissue.
  4. Block plates. Add 150 µl of blocking buffer/well, cover plate, and incubate at 37°C for 2 hr.
  5. After blocking, wash plate (Section C, Step 3). Plate is ready to use.

D. Test Procedure

  1. Lysates can be used undiluted or diluted in blocking buffer. 100 µl of lysate is added per well. Cover plate and incubate at 37°C for 2 hr.
  2. Wash plate (Section C, Step 3).
  3. Dilute detection antibody 1:100 in blocking buffer. For a single 96 well plate, add 100 µl of detection antibody Stock to 9.9 ml of blocking buffer. Mix well and add 100 µl/well. Cover plate and incubate at 37°C for 1 hr.
  4. Wash plate (Section C, Step 3).
  5. Secondary antibody, either streptavidin anti-mouse or anti-rabbit-HRP, is diluted 1:1000 in blocking buffer. For a single 96 well plate, add 10 µl of secondary antibody stock to 9.99 ml of blocking buffer. Mix well and add 100 µl/well. Cover and incubate at 37°C for 30 min.
  6. Wash plate (Section C, Step 3).
  7. Add 100 µl of TMB substrate per well. Cover and incubate at 37°C for 10 min.
  8. Add 100 µl of STOP solution per well. Shake gently for a few seconds.
  9. Read plate on a microplate reader at absorbance 450 nm.
    1. Visual Determination: Read within 30 min after adding STOP solution.
    2. Spectrophotometric Determination: Wipe underside of wells with a lint-free tissue. Read absorbance at 450 nm within 30 min after adding STOP solution.

posted January 2008

revised Sepetember 2013

Product Includes Volume Cap Color
β-Actin Capture Rabbit mAb (100X) 400 µl Pink
Pan-Actin Detection Mouse mAb (100X) 400 µl Blue
Anti-mouse IgG, HRP-linked Antibody (1000X) 40 µl Yellow

CST's PathScan® Total β-Actin Sandwich ELISA Antibody Pair is offered as an economical alternative to our PathScan® Total β-Actin Sandwich ELISA Kit #7880. Capture and detection antibodies (100X stocks) and an HRP-linked secondary antibody (1000X stock) are supplied. Sufficient reagents are supplied for 4 x 96 well ELISAs. The β-actin rabbit capture antibody is coated in PBS overnight onto a 96 well microplate. After blocking, cell lysate is added followed by pan-actin mouse detection antibody and HRP-linked, anti-mouse IgG antibody. HRP substrate,TMB, is then added for color development. 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.

Storage:

Capture and detection antibodies are stored at 4°C. HRP-linked secondary reagent is stored at -20°C.

For Antibody Pair specificity and sensitivity, please refer to the corresponding PathScan® Sandwich ELISA Kit. Note: This antibody pair detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.

Species Reactivity:

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 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, 288-93.
  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.
Entrez-Gene Id
60
Swiss-Prot Acc.
P60709
For Research Use Only. Not For Use In Diagnostic Procedures.

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
PathScan is a trademark of Cell Signaling Technology, Inc.
U.S. Patent No. 7,429,487, foreign equivalents, and child patents deriving therefrom.

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7881S		 1 Kit  (Reagents for 4 x 96 well plates) N/A

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