Mouse monoclonal anti ha antibody 12ca5

Isoelectric focusing of yeast extracts was adapted from a previously described method^{15 (link)}. Briefly, yeast cell cultures cells were lysed in buffer A (10% glycerol, 50 mM Tris HCl pH 7.5, 150 mM NaCl, 0.1% Nonidet NP-40, plus specific proteases inhibitor, Sigma Chemical). The lysate was cleared by centrifugation for 10 min at 13,000 rpm at 4 °C, and protein concentration was determined. Equal amounts of protein, generally 1 mg, were precipitated. Protein pellets were resuspended in rehydration buffer (8 M urea, 1% Chaps, 50 mM DTT, 0.2% biolytes (Bio-Rad, Bio-Lyte 3/10), 0.001% bromophenol blue), and samples were loaded onto Bio-Rad ReadyStrip IPG strips (11 cm, pH 5–8) for separation. Following isoelectric focusing, proteins were resolved in SDS-PAGE gels and transferred to Hybond-ECL membranes. Rho1-HA and GFP-Rho2-HA fusions were detected by immunoblot analysis with a mouse monoclonal anti-HA antibody (12CA5, Roche Molecular Biochemicals). Cdc42-GFP^SW and GFP-Ras1 fusions were detected with mouse monoclonal anti-GFP antibody (Roche). FLAG-Ryh1 fusion was detected with mouse monoclonal anti-FLAG antibody (Sigma-Aldrich). Immunoreactive bands were revealed with anti-mouse-HRP-conjugated secondary antibodies (Sigma-Aldrich) and the ECL system (GE-Healthcare).

Preparation of cell extracts, affinity chromatography purification of HA-tagged Pmk1 or Sty1 with Ni²⁺-NTA agarose beads (Qiagen), and SDS-PAGE were performed as described previously (32 (link)). This approach strongly reduces the potential inaccuracy in the detection of both total and phosphorylated MAPKs. Dual phosphorylation in either Pmk1 or Sty1 was detected by employing rabbit polyclonal anti-phospho-p44/42 (Cell Signaling) or rabbit monoclonal anti-phospho-p38 (Cell Signaling) antibody, respectively. Total Pmk1 or Sty1 was detected with mouse monoclonal anti-HA antibody (12CA5; Roche Molecular Biochemicals). Immunoreactive bands were revealed with anti-rabbit or anti-mouse horseradish peroxidase (HRP)-conjugated secondary antibodies (Sigma-Aldrich) and the ECL system (GE Healthcare).

In experiments carried out with budding yeast, immunodetection of Glucose-6-phosphate dehydrogenase (G6PDH) and Myc-tagged proteins was carried out using polyclonal anti-G6PDH (Sigma) and monoclonal 4A6 (Millipore) or 9E10 (Santa Cruz Biotechnology) antibodies, respectively. Polyclonal anti-phospho-p44/p42 MAPK (Thr202/Tyr204) (Cell Signaling), anti-GST (Santa Cruz Biotechnology) and anti-His antibodies (Sigma) were also used as described previously [18] (link). The primary antibodies were detected using a fluorescently-conjugated secondary antibody with an Odyssey Infrared Imaging System (LI-COR Biosciences).
In experiments performed with fission yeast, dual phosphorylation in Pmk1 was detected with polyclonal anti-phospho-p42/p44 as above, whereas total Pmk1 was detected after incubation with mouse monoclonal anti-HA antibody (12CA5, Roche Molecular Biochemicals).The immunoreactive bands were revealed with an anti-mouse-HRP-conjugated secondary antibody (Sigma) and the ECL system (GE Healthcare). GST fusions were detected with a goat anti-GST-HRP conjugated polyclonal antibody (GE Healthcare).