Phospho-ERK1/ERK2 (p44/p42 MAPK) (T202/Y204)
| Cat Number: | ABP-83922 |
|---|---|
| Conjugate: | Unconjugated |
| Size: | 100 ug |
| Clone: | Poly |
| Concentration: | 1mg/ml |
| Host: | Rb |
| Isotype: | IgG |
| Immunogen: | Peptide derived from the protein area including conserved pT-E-pY motif of activated Erk 1,2 |
| Reactivity: | Hu, Ms, Rt |
| Applications: | Western blotting: 1:1000 Immunohistochemistry: 1:100-500 Immunofluorescence: 1:100-500 Immunoprecipitation: to be determined Immunocytochemistry: 1:100 |
| Form: | liquid |
| Buffer: | 20 mM Tris Hcl, pH 8 10 mg/mL BSA - 0,05% Sodium Azide |
| Storage: | The antibody can be stored at 2° - 8° C for 1 month without detectable loss of activity. Antibody can also be aliquotted and stored frozen at -20° C to -70° C in a manual defrost freezer for six months without detectable loss of activity. Avoid repeated freeze-thaw cycles. |
Detection of ERK1 and ERK2 phosphorylated at T202/Y204 and T185/Y187, respectively. Human HeLa cells were incubated with 200 nM PMA for the indicated times. Total cell lysates in gel sample buffer were resolved by SDS-PAGE.
WESTERN BLOT (WB ) PROTOCOL
Western immunoblotting solutions:
Wash buffer: 1x Tris Buffered Saline (TBS); 0.1% Triton X-100 –
Blocking buffer: 1xTBS; 0.1% Triton X-100; 5% BSA (used with the primary antibody)
For western blots, incubate the membrane with antibody diluted in blocking buffer 2 hours at room temperature
IMMUNOCYTOCHEMISTRY (ICC) PROTOCOL - INSTRUCTION MANUAL
1. Coat coverslips with 1% gelatin-coating solution for 2 hours at room temperature (RT); rinse with distilled water, and let to dry overnight. Before plating the cells, wash the coated coverslips briefly with PBS. 2. Fix the cells with 4% paraformaldehyde solution (in PBS, pH 7.2), for 15 min at RT. 3. Wash 2 x 3 min with PBS. 4. Permeabilize the cells with 0.1% Triton X-100 solution (in PBS, pH 7.2) for 5 min on ice. 5. Wash 2 x 3 min with PBS. 6. Incubate the cells in blocking buffer (0.3M glycine in PBS, 2% BSA) for 30 min at RT. 7. Incubate the cells with primary antibody: anti-phospho Erk 1,2 clonal antibody at the dilution of 1:100 – in antibody dilution buffer (PBS, 1% BSA) for 1 hour at RT in humid chamber. 8. Wash 2 x 3 min with PBS. 9. Apply the secondary antibody used at 1:300 in antibody dilution buffer, and cells were incubated for 1 hour at RT in dark). 10. Wash 3 x 3 min with PBS. 11. Rinse once with distilled water. 12. Mount the slide for observation, with a drop of anti-fade mounting medium.