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® Phospho-MARCKS (Ser152/156) Sandwich ELISA Kit is shown.
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. 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.
|Product Includes||Volume||Solution Color|
|MARCKS Rabbit mAb Coated Microwells||96 tests|
|Phospho-MARCKS (Ser152/156) Rabbit Detection Antibody (Biotinylated)||1 ea||Green (Lyophilized)|
|HRP-Linked Streptavidin (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 Sample Diluent||25 ml||Blue|
|ELISA Wash Buffer (20X) 9801||25 ml|
|PathScan® Sandwich ELISA Lysis Buffer (1X) 7018||30 ml|
The PathScan® Phospho-MARCKS (Ser152/156) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of MARCKS when phosphorylated at Serines 152 and 156. A MARCKS rabbit antibody has been coated onto the microwells. After incubation with cell lysates, MARCKS protein (phosphorylated and nonphospho) is captured by the coated antibody. Following extensive washing, a biotinylated phospho-MARCKS (Ser152/156) rabbit monoclonal detection antibody is added to detect the captured phospho-MARCKS protein. HRP-linked streptavidin is then used to recognize the bound detection antibody. HRP substrate, TMB, is added to develop color. The magnitude of the absorbance for the developed color is proportional to the quantity of MARCKS phosphorylated at Serines 152 and 156.
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
revised January 2016
Protocol Id: 205
PathScan® Phospho-MARCKS (Ser152/156) Sandwich ELISA Kit #7251 detects endogenous levels of MARCKS protein only when phosphorylated at Serines 152 and 156 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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