The procedure presented below describes a facile method for studying signal transduction events with adherent cells (HeLa, MCF-7, BALB/c 3T3, etc.). In this procedure, cells are gently lifted from their culture vessel, placed into wells of a 96-well filter bottom plate, and stimulated as desired. At the end of stimulation, cell culture medium is removed from the bottom of the wells by gentle aspiration using a vacuum manifold. The cells are then washed with PBS, aspirated, and lysed within the wells by addition of Cell Extraction Buffer. The cell extracts are then assayed using Invitrogen p38MAPK [pTpY180/182] and p38MAPK (total) phosphoELISA kits.
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Add Protease Inhibitor Cocktail (Cat. No. ) directly to the lysis buffer or extract to produce a 1X final working solution (10 µL of Halt Protease Inhibitor Cocktail per mL of cell lysis buffer).
Figure 2 shows the results obtained with p38 MAPK [pTpY180/182] phosphoELISA (Cat. No. KHO). The data presented show that anisomycin treatment increases the level of phosphorylation of p38 MAPK at threonine 180 and tyrosine 182, and that this is directly proportional to the number of cells seeded.
HeLa cells were seeded into the wells of the plate at densities of 3,000, 6,000, and 12,000 cells in 200 μL cell culture media. The seeded cells were incubated for 18 hours at 37°C, then treated with anisomycin (100 μM) for 60 minutes, or left untreated. At the end of the incubation period, the cells were lysed by the method described above, and assayed with the p38 MAPK [pTpY180/182] phosphoELISA kit.
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The procedure presented below describes a facile method for studying signal transduction events with suspension cells (Jurkat, Raji, THP-1, etc.). In this procedure, cells are plated into the wells of 96-well filter bottom plates and stimulated as desired. At the end of the stimulation, cell culture medium is removed from the bottom of the wells by gentle aspiration using a vacuum manifold. The cells are then washed with PBS, aspirated, and lysed within the wells by addition of Cell Extraction Buffer. The cell extracts are then assayed using Invitrogen p38MAPK [pTpY180/182] and p38MAPK (total) phosphoELISA&#; kits.
The results presented in Figure 1 were obtained with the p38 MAPK [pTpY180/182] phosphoELISA&#; (Cat. No. KHO). The data clearly demonstrates that anisomycin treatment increases the level of phosphorylation of p38 MAPK at threonine 180 and tyrosine 182. The data presented in Figure 1 also show that the quantity of p38 MAPK [pTpY180/182] measured with this kit is directly proportional to the number of cells seeded into the wells of the filter bottom plate. The results presented in Figure 2 were obtained with the p38 MAPK Total phosphoELISA&#; (Cat. No. KHO). This kit was designed to permit normalization of p38 MAPK phosphorylation state. The data presented in Figure 2 show that anisomycin treatment has no impact on the amount of total p38 MAPK measured in cell lysates, indicating anisomycin increases p38 MAPK [pTpY180/182] by enhancing phosphorylation state (Figure 1), rather than altering total p38 MAPK level. The data presented also show that the quantity of total p38 MAPK measured with this kit is directly proportional to the number of cells seeded into the wells of the filter bottom plate.
Figure 2. p38 MAPK [pTpY180/182] phosphoELISA&#; kit. Anisomycin treatment of Jurkat cells does not change the level of p38 MAPK (Total) expression.
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