REACTIVITY | H M |
Product Information
NOTE: Refer to product-specific datasheets or product webpage for assay incubation temperature.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
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.
NOTE: Refer to product-specific datasheet or webpage for lysis buffer recommendation.
Add 100 µl of STOP solution to each well. Shake gently for a few seconds.
NOTE: Initial color of positive reaction is blue, which changes to yellow upon addition of STOP solution.
posted June 2005
revised November 2013
Protocol Id: 21
Both p44 and p42 MAP kinases (Erk1 and Erk2) function in a protein kinase cascade that plays a critical role in the regulation of cell growth and differentiation (1-6). MAP kinases are activated by a wide variety of extracellular signals including growth and neurotrophic factors, cytokines, hormones and neurotransmitters. Activation of MAP kinases occurs through phosphorylation of threonine and tyrosine (Thr202 and Tyrr204 of human MAP kinase or Thr183 and Tyr185 of rat MAP kinase) at the sequence T*EY* by a single upstream MAP kinase kinase (MEK) (7,8).
MEK1 and MEK2 are dual-specificity protein kinases that function in a mitogen activated protein kinase cascade controlling cell growth and differentiation. Activation of MEK1 and MEK2 occurs through phosphorylation of Ser217 and Ser221 by Raf-like molecules. MEK activates p44 and p42 MAP kinase (1,9,10).
p38 MAP kinase (MAPK) participates in a signaling cascade controlling the cellular response to pro-inflammatory cytokines and a variety of cellular stresses. MKK3, MKK6 and SEK (MKK4) activate p38 MAP kinase by phosphorylation at Thr180 and Tyr182 (11-14).
The stress-activated protein kinase/Jun-amino-terminal kinase SAPK/JNK is activated by a variety of environmental stresses, including UV and gamma radiation, ceramides, inflammatory cytokines and in some instances, by growth factors and GPCR agonists (15-20). As with the other MAPKs, the core-signaling unit is composed of a MAPKKK, typically MEKK1-4, or by a mixed lineage kinase (MLK), which phosphorylates and activates MKK4-7, which then phosphorylates Thr183 and Tyr185 to activate the SAPK/JNK kinase (16). Stress signals are delivered to this cascade by small GTPases of the Rho family (Rac, Rho, cdc42) (17). Both Rac1 and cdc42 mediate the stimulation of MEKKs and MLKs (17). Alternatively, MKK4-7 can be activated by a pathway independent of small GTPases via stimulation of a member of the germinal center kinase (GCK) family (18).
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