Cell Signaling Technology

Product Pathways - Ca / cAMP / Lipid Signaling

PathScan® Total MARCKS Sandwich ELISA Kit #7188

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Kit Includes Volume Solution Color
MARCKS Mouse Ab Coated Microwells 96 tests
MARCKS Rabbit Detection Ab 11 ml Green
Anti-Rabbit 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
PathScan® Sandwich ELISA Lysis Buffer (1X) #7018 30 ml Colorless

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

Reactivity Key:  H=Human
Species enclosed in parentheses are predicted to react based on 100% sequence homology.

Description

The PathScan® Total MARCKS Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of MARCKS protein. A MARCKS mouse antibody has been coated onto the microwells. After incubation with cell lysates, MARCKS (phospho and nonphospho) is captured by the coated antibody. Following extensive washing, a MARCKS rabbit detection antibody is added to detect captured MARCKS protein. Anti-Rabbit 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 total MARCKS protein.Antibodies in kit are custom formulations specific to kit.

Specificity / Sensitivity

PathScan® Total MARCKS Sandwich ELISA Kit #7188 detects endogenous levels of MARCKS protein as shown in Figure 1. Kit sensitivity is shown in Figure 2. This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.

ELISA

ELISA

Figure 2. The relationship between the protein concentration of lysates from untreated, bisindolylmaleimide-treated or TPA-treated HeLa cells and the absorbance at 450 nm using the PathScan® Total MARCKS Sandwich ELISA Kit is shown.

ELISA - Western correlation

ELISA - Western correlation

Figure 1. Treatment of HeLa cells with TPA #4174 stimulates phosphorylation of MARCKS at Ser152/Ser156, while cells treated with bisindolylmaleimide inhibits phosphorylation, as detected by the PathScan® Phospho-MARCKS (Ser152/156) Sandwich ELISA Kit #7251. The total level of MARCKS is not affected by either treatment as detected by PathScan® Total MARCKS Sandwich ELISA Kit #7188. HeLa cells (80-90% confluent) were treated with 200 μM TPA for 30 minutes at 37ºC, or treated with 2.0 μM bisindolylmaleimide for 3 hours. The absorbance readings at 450 nm are shown in the top figure, while the corresponding western blots using MARCKS (D88D11) XP® Rabbit mAb #5607 (left panel) or Phospho-MARCKS (Ser152/156) Antibody #2741 (right panel) are shown in the bottom figure.

Background

Myristoylated Alanine-Rich C-Kinase Substrate (MARCKS) is a major PKC substrate expressed in many cell types. MARCKS has been implicated in cell motility, cell adhesion, phagocytosis, membrane traffic, and mitogenesis (1). PKC phosphorylates Ser159, 163, 167, and 170 of MARCKS in response to growth factors and oxidative stress. Phosphorylation at these sites regulates the calcium/calmodulin binding and filamentous (F)-actin cross-linking activities of MARCKS (2-4). Phosphorylation by PKC also results in translocation of MARCKS from the plasma membrane to the cytoplasm (5).

  1. Ramsden, J.J. (2000) Int J Biochem Cell Biol 32, 475-9.
  2. Heemskerk, F.M. et al. (1993) Biochem Biophys Res Commun 190, 236-41.
  3. Graff, J.M. et al. (1989) J Biol Chem 264, 21818-23.
  4. Hartwig, J.H. et al. (1992) Nature 356, 618-22.
  5. Thelen, M. et al. (1991) Nature 351, 320-2.

Application References

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

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