Description
The members of the Mitogen-Activated Protein Kinase (MAPK) family are components of a key signal transduction cascade that links events at the cell surface to responses in the nucleus. The signaling cascade is found in species as varied as yeast and humans, with many of the proteins being well conserved. In mammals the most widely studied members of the cascade are the Extracellular signal-Regulated Kinases, ERK1 (p44 MAPK) and ERK2 (p42 MAPK). ERK1 and ERK2 share 85% homology and are activated by extracellular signals such as growth factors, hormones, and phorbol esters. Activation occurs through a series of phosphorylations by kinases activating other kinases and eventually leading to phosphorylation of the ERKs. Growth factor stimulation leads to activation of Ras and Raf, leading to phosphorylation of MEK1 (MAPK/ERK kinase) which, in turn, activates the ERKs via dual phosphorylation. Once activated, the ERKs phosphorylate other cytoplasmic signalling molecules, cell-surface receptors, microtubule-associated proteins, and transcription factors in the nucleus. Thus, the active ERK has myriad downstream effectors that implicate it in the control of cell proliferation and differentiation, as well as regulation of the cytoskeleton. Furthermore, studies have shown that elevated ERK activity is associated with some cancers. The 20A monoclonal antibody recognizes the phosphorylated threonine 202 and tyrosine 204 (pT202/pY204) of human ERK1 and pT184/pY186 of human ERK2. The orthologous phosphorylation sites in murine ERK1 and ERK2 are T203/Y205 and T183/Y185.
Bioimaging 1. Seed the cells in appropriate culture medium at ~10,000 cells per well in a Falcon® 96-well Imaging Plate (Cat. No. 353219) and culture overnight. 2. Remove the culture medium from the wells, and fix the cells by adding 100 μl of BD Cytofix™ Fixation Buffer (Cat. No. 554655) to each well. Incubate for 10 minutes at room temperature (RT). 3. Remove the fixative from the wells, and permeabilize the cells using either BD Perm Buffer III, 90% methanol, or Triton™ X-100: a. Add 100 μl of -20°C 90% methanol or Perm Buffer III (Cat. No. 558050) to each well and incubate for 5 minutes at RT. OR b. Add 100 μl of 0.1% Triton™ X-100 to each well and incubate for 5 minutes at RT. 4. Remove the permeabilization buffer, and wash the wells twice with 100 μl of 1× PBS. 5. Remove the PBS, and block the cells by adding 100 μl of BD Pharmingen™ Stain Buffer (FBS) (Cat. No. 554656) to each well. Incubate for 30 minutes at RT. 6. Remove the blocking buffer and add 50 μl of the optimally titrated primary antibody (diluted in Stain Buffer) to each well, and incubate for 1 hour at RT. 7. Remove the primary antibody, and wash the wells three times with 100 μl of 1× PBS. 8. Remove the PBS, and add the second step reagent at its optimally titrated concentration in 50 μl to each well, and incubate in the dark for 1 hour at RT. 9. Remove the second step reagent, and wash the wells three times with 100 μl of 1× PBS. 10. Remove the PBS, and counter-stain the nuclei by adding 200 μl per well of 2 μg/ml Hoechst 33342 (e.g., Sigma-Aldrich Cat. No. B2261) in 1× PBS to each well at least 15 minutes before imaging. 11. View and analyze the cells on an appropriate imaging instrument.
Store undiluted at -20°C. The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography.
1. Since applications vary, each investigator should titrate the reagent to obtain optimal results.
2. This antibody has been developed and certified for the bioimaging application. However, a routine bioimaging test is not performed on every lot. Researchers are encouraged to titrate the reagent for optimal performance.
3. Triton is a trademark of the Dow Chemical Company.
4. Source of all serum proteins is from USDA inspected abattoirs located in the United States.
5. Caution: Sodium azide yields highly toxic hydrazoic acid under acidic conditions. Dilute azide compounds in running water before discarding to avoid accumulation of potentially explosive deposits in plumbing.
6. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
