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.
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.
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.
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).
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