For the Mkc1p phosphorylation blot, overnight cultures were diluted in fresh YEPD media to an OD600 of 0.3 and grown until they reached an OD600 of 1 at 37°C and 200 rpm. Samples were treated with 5 μg ml−1 tunicamycin for 2 hr before they were recovered. The procedures used for cell collection, lysis, protein extraction, fractionation by SDS-PAGE and transfer to nitrocellulose membranes have been previously described (Martín, Arroyo, Sánchez, Molina, & Nombela, 1993 (link)). Anti-phosphop44/42 MAP kinase (Thr202/Tyr204) antibody (Anti-p42–44-P) (Cell Signalling Technology, Inc.) was used to detect dually phosphorylated Mkc1 and Cek1 MAPKs, and polyclonal anti-Mkc1 antibodies were used for Mkc1 detection (Federico Navarro-García, Eisman, Fiuza, Nombela, & Pla, 2005 (link)). Blot imaging was done by using an Odyssey fluorescence imager (LI-COR) and quantified using Image Studio Lite (LI-COR).
Anti phosphop44 42 map kinase thr202 tyr204 antibody anti p42 44 p
Manufactured by Cell Signaling Technology
The Anti-phospho-p44/42 MAP kinase (Thr202/Tyr204) antibody (Anti-p42-44-P) is a protein-specific antibody that recognizes the phosphorylated forms of p44/42 MAPK (Erk1/2) at Thr202 and Tyr204 residues. It is used for the detection and analysis of the activated, phosphorylated state of these MAPK proteins.
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2 protocols using anti phosphop44 42 map kinase thr202 tyr204 antibody anti p42 44 p
1
Mkc1p Phosphorylation Monitoring in Yeast
2
Mkc1p Phosphorylation Blotting Protocol
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For the Mkc1p phosphorylation blot, overnight cultures were diluted in fresh YEPD media to an OD600 of 0.3 and grown until they reached an OD600 of 1 at 37°C and 200 rpm. Samples were treated with 5 μg ml−1 tunicamycin for 2 hr before they were recovered. The procedures used for cell collection, lysis, protein extraction, fractionation by SDS‐PAGE and transfer to nitrocellulose membranes have been previously described (Martín, Arroyo, Sánchez, Molina, & Nombela, 1993 ). Anti‐phospho‐p44/42 MAP kinase (Thr202/Tyr204) antibody (Anti‐p42‐44‐P) (Cell Signalling Technology, Inc.) was used to detect dually phosphorylated Mkc1 and Cek1 MAPKs, and polyclonal anti‐Mkc1 antibodies were used for Mkc1 detection (Federico Navarro‐García, Eisman, Fiuza, Nombela, & Pla, 2005 ). Blot imaging was done by using an Odyssey fluorescence imager (LI‐COR) and quantified using Image Studio Lite (LI‐COR).
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