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. Author manuscript; available in PMC: 2010 Nov 20.
Published in final edited form as: ACS Chem Biol. 2009 Nov 20;4(11):958–968. doi: 10.1021/cb9002255

Figure 2. Validation of uH2BG76A as a surrogate for uH2B.

Figure 2

a, Reversed phase high performance liquid chromatography (RP-HPLC) chromatogram of purified uH2BG76A, 7. b, Electrospray ionization mass spectrometry (ESI-MS) spectrum of purified uH2BG76A, 7. Charge states are labelled. [(M+H)+ observed = 22,380 ± 4 Da (s.d.). (M+H)+ expected = 22,379 Da.] c, UCH-L3-mediated hydrolysis of uH2B and uH2BG76A. Coomassie stained gel of assay samples quenched after indicated times. + indicates addition of UCH-L3 at 0 min; ++ indicates addition of UCH-L3 at 0 and 10 min. d, Western blot of uH2B and uH2BG76A with linkage specific α-uH2B antibody (top panel). Western blot with α-ubiquitin antibody (middle panel) and ponceau stain (bottom panel) represent loading controls. e, Dot1L methyltransferase assay on uH2B and uH2BG76A containing nucleosomes. Nucleosomes methylated with 3H S-adenosyl methionine (SAM) were separated on native gels and stained with ethidium bromide (middle panel) prior to probing for 3H methyl incorporation by fluorography (top panel). Quantification of methyltransferase activity was performed by filter-binding followed by liquid scintillation counting (bottom panel). Error bars represent one s.d. (n = 3).