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. Author manuscript; available in PMC: 2013 Mar 28.
Published in final edited form as: Methods. 2011 Feb 15;54(3):296–303. doi: 10.1016/j.ymeth.2011.02.003

Figure 2. Detection and enrichment of modified Flag-H2B from TCA extracts.

Figure 2

(A) 30 mg of total protein from a TCA extract of cells expressing Flag-tagged H2B were electrophoresed on a 15 % SDS polyacrylamide gel and transferred to an Immobilon-P membrane. Unmodified and ubiquitylated H2B were detected by Western blot analysis using anti-Flag antibodies. (B) 150 mg of the TCA extract in (A) were enriched on M2 agarose beads, and a portion of the eluate was electrophoresed on a 15 % SDS polyacrylamide gel and subjected to Western blot analysis. The left panel was probed with anti-Flag antibody and the right panel was blotted with antibodies raised against recombinant GST-Smt3 (a gift from Pam Meluh). (C) TCA extracts derived from a strain expressing Flag-H2B and containing a 6xHis-ubiquitin gene driven by a CUP1 promoter (in addition to the endogenous ubiquitin genes) were purified on either M2 agarose beads (left panel) or a Talon metal affinity resin (right panel). Western blot analysis using anti-Flag antibodies was performed to detect the modified Flag-H2B species. * Non-specific bands