Journal: Methods and Protocols

Article Title: Quantitative Assessment of Histone H2B Monoubiquitination in Yeast Using Immunoblotting

doi: 10.3390/mps5050074

Figure Lengend Snippet: Workflow for the quantitative assessment of histone H2B monoubiquitination using immunoblot.

Article Snippet: Unlike with the S. cerevisiae extracts ( and ), the anti-yeast histone H2B antibody (Active Motif; 1:1000) yielded high background or cross-reactivity when applied to the S. pombe extracts ( a, first panel).

Techniques: Western Blot

Journal: Methods and Protocols

Article Title: Quantitative Assessment of Histone H2B Monoubiquitination in Yeast Using Immunoblotting

doi: 10.3390/mps5050074

Figure Lengend Snippet: Detection of H2BK123ub1 in the indicated wild-type (WT) and mutant S. cerevisiae strains.( a ) Entire membrane probed with anti-yeast H2B (Active Motif) antibody (1:1000 dilution). ( b ) Sections of membrane probed with anti-yeast H2B (Active Motif) antibody at 1:1000 dilution (upper blot) and 1:10,000 (lower blot). Black arrow , H2BK123 monoubiquitinated form. Grey arrow, diubiquitinated form of H2B. ( c ) Section of membrane probed with anti-ubiquityl-H2B (K120) (Cell Signaling Technology) antibody (1:1000). Proteins stained with Ponceau S serve as loading controls. Molecular weights (kDa) based on protein size makers are indicated to the left of each blot. Asterisk, unspecific cross-reacting protein.

Article Snippet: Unlike with the S. cerevisiae extracts ( and ), the anti-yeast histone H2B antibody (Active Motif; 1:1000) yielded high background or cross-reactivity when applied to the S. pombe extracts ( a, first panel).

Techniques: Mutagenesis, Membrane, Staining

Journal: Methods and Protocols

Article Title: Quantitative Assessment of Histone H2B Monoubiquitination in Yeast Using Immunoblotting

doi: 10.3390/mps5050074

Figure Lengend Snippet: Antibody dilutions used.

Article Snippet: Unlike with the S. cerevisiae extracts ( and ), the anti-yeast histone H2B antibody (Active Motif; 1:1000) yielded high background or cross-reactivity when applied to the S. pombe extracts ( a, first panel).

Techniques:

Journal: Methods and Protocols

Article Title: Quantitative Assessment of Histone H2B Monoubiquitination in Yeast Using Immunoblotting

doi: 10.3390/mps5050074

Figure Lengend Snippet: ( a ) Sections of membrane probed with the indicated antibodies to quantify histones H3 or H2B and H2BK123ub1 levels in wild-type (WT) and indicated deubiquitinase mutant S. cerevisiae strains. The antibody dilutions used are listed in . ( b ) Quantitation of histones H3 (i) and H2BK123ub1 (ii) levels normalized to Ponceau S stained proteins from two or three independent experiments, respectively. ( c ) Sections of membrane probed with anti-H2B antibody (1:1000 dilution) (upper), anti-ubiquityl H2BK120 antibody (1:1000) (middle), and stained with Ponceau S (lower) using extracts prepared from S. cerevisiae strains expressing untagged or C-terminal V5 epitope-tagged histone H2B.

Article Snippet: Unlike with the S. cerevisiae extracts ( and ), the anti-yeast histone H2B antibody (Active Motif; 1:1000) yielded high background or cross-reactivity when applied to the S. pombe extracts ( a, first panel).

Techniques: Membrane, Mutagenesis, Quantitation Assay, Staining, Expressing

Journal: Methods and Protocols

Article Title: Quantitative Assessment of Histone H2B Monoubiquitination in Yeast Using Immunoblotting

doi: 10.3390/mps5050074

Figure Lengend Snippet: ( a ) Samples from extracts prepared from S. pombe strains expressing untagged H2B or Flag-tagged H2B were spearated and transferred onto a PVDF membrane. Shown are whole or sections of membrane probed with anti-histone H2B antibody from Active Motif and from Gene Tex and with anti-H2BK120 ubiquityl antibody. Ponceau S dye-stained sections used for normalization are also shown. Asterisk indicates cross-reacting proteins. ( b ) Samples from extracts prepared from wild-type S. pombe or strains lacking Ubp8 or Brl1 were separated and transferred to PVDF membrane. Shown are sections of membrane probed with anti-H2B (Gene Tex) or anti-H2BK120 ubiquityl antibody are shown. A section of the membrane stained with Ponceau S used for normalization is also shown. ( c ) Quantitation of H2BK119ub1 levels in the S. pombe ubp8-null mutant relative to wild-type (WT) strain. Error bars denote standard deviation of the mean from three independent experiments.

Article Snippet: Unlike with the S. cerevisiae extracts ( and ), the anti-yeast histone H2B antibody (Active Motif; 1:1000) yielded high background or cross-reactivity when applied to the S. pombe extracts ( a, first panel).

Techniques: Expressing, Membrane, Staining, Quantitation Assay, Mutagenesis, Standard Deviation