Ha tag 6e2 mouse monoclonal antibody

All chemicals used were obtained from commercial sources and were of analytical grade. Media components, fine chemicals and reagents were purchased from Sigma Aldrich (St. Louis, USA), HiMedia (Mumbai, India), Merck Millipore India Ltd (Mumbai, India), USB Corporation (Ohio, USA) or Difco, USA. Cysteine and cystine were also purchased from Sigma Aldrich (St. Louis, USA). HA-Tag (6E2) Mouse monoclonal antibody and horse anti-mouse HRP-linked antibody were bought from Cell Signaling Technology (Danvers, MA, USA). Alexa Flour^® 488 conjugated goat anti-mouse antibody was obtained from Molecular probes (Eugene, Oregon, USA). Cysteine stock solutions that were prepared fresh were made by dissolving the required amount of Cysteine in 1 ml of de-ionized water, which was then filter-sterilized using 0.2 μ filter membrane. cystine stock solutions were prepared by dissolving the required amount of cystine in 1 ml of deionized water along with 25 µL of concentrated HCl, filter-sterilized using 0.2 μ filter membrane. ¹⁴C cystine and custom synthesized ³⁵S cystine was procured from Perkin Elmer Ltd. (Boston, USA).

Total protein extracts were prepared, then analysed for levels of Hsp90 and Slt2 MAP kinase essentially as described previously (Millson et al. 2005 (link); Truman et al. 2006 (link), 2007 (link)). The antisera used were as follows: for the analysis of dually phosphorylated (Thr¹⁹⁰/Tyr¹⁹²)-Slt2, a commercial antibody raised against dually phosphorylated (Thr²⁰²/Tyr²⁰⁴)-p44/42 MAP kinase (New England Biolabs) that recognises the dually phosphorylated (Thr¹⁹⁰/Tyr¹⁹²)-Slt2 MAP kinase in yeast cell extracts (Martin et al. 2000 (link)); for Slt2-3xHA analysis, a HA-Tag (6E2) mouse monoclonal antibody (Cell Signalling); for Hsp82-His₆ analysis, a monoclonal anti-tetraHis antibody (Qiagen). Slt2-HA was isolated by immunoprecipitation using anti-HA immunoglobulin G-coated agarose beads (Sigma).

All the chemicals used in this study were analytical grade and obtained from commercial sources. Media components were purchased from Difco (Detroit, MI) Sigma Aldrich, (St. Louis, MO), HiMedia, (Mumbai, India), Merck India Ltd (Mumbai, India), and USB Corporation (Cleveland, OH). Oligonucleotides were purchased from Sigma India. Restriction enzymes, Vent DNA polymerase, and other DNA-modifying enzymes were obtained from New England Biolabs (Beverly, MA). DNA sequencing kit (ABI PRISM 310 XL with dye termination cycle sequencing ready reaction kit) was obtained from Perkin Elmer, (Norwalk, CT). Gel-extraction kits and plasmid miniprep columns were obtained from QIAGEN (Valencia, CA) or Sigma (St. Louis, MO). [³⁵S] GSH (specific activity 1000 Ci mmol^-1) was purchased from Bhabha Atomic Research Centre, Mumbai, India. HA-Tag (6E2) mouse monoclonal antibody and horse anti-mouse HRP-linked antibody were bought from Cell Signaling (Danvers, MA). Alexa Flour 488 conjugated goat anti-mouse antibody was obtained from Molecular Probes (Eugene, OR). Hybond ECL (nitrocellulose) membrane and ECL plus Western blotting detection reagents were purchased from Amersham Biosciences (UK).