flag antibody-bound magnetic beads  (CH Instruments)

Journal:

Article Title: Schizosacchromyces pombe Dpb2 Binds to Origin DNA Early in S Phase and Is Required for Chromosomal DNA Replication

doi: 10.1091/mbc.E03-02-0088

Figure Lengend Snippet: 4xFLAG-Dpb2 binds ARS elements early in S phase. (A) Analysis of DNA content by flow cytometry indicates that DNA synthesis begins at 60–80 min after release from the G2 block. (B) Relative enrichment ratio of immunoprecipitated ARS DNA to nonARS DNA was calculated and plotted from the raw data in C. (C and D) ChIP analyses with antibody to the FLAG-epitope for Dpb2 (C) or antibody to Mcm6 (D) were used to measure the levels of Dpb2 and Mcm6 at ARS loci. DNA isolated from the immunoprecipitated chromatin (IP) or from whole cell soluble extracts (Total DNA) was subjected to PCR to amplify DNA fragments from either ars2004, ars3002 or from nonARS DNA. Please note that the peak of Dpb2 binding to ARS elements occurs 60 min after release from the G2 block (C) in contrast to Mcm6 binding which peaks at 40 min postrelease (D). No significant chromatin binding was observed when cross-linking agent was omitted, if cell lysates were prepared in the absence of FLAG-tagged protein, or if antibodies were excluded from the immunoprecipitation reaction (our unpublished data).

Article Snippet: The lysate was then clarified by centrifugation (12,000 g , 15 min) at 4°C, and 90% of the supernatant was incubated with anti-FLAG or anti-Mcm6p antibodies bound to magnetic beads (Dynal Biotech, Oslo, Norway).

Techniques: Flow Cytometry, DNA Synthesis, Blocking Assay, Immunoprecipitation, FLAG-tag, Isolation, Chromatin Immunoprecipitation, Binding Assay

Journal:

Article Title: Schizosacchromyces pombe Dpb2 Binds to Origin DNA Early in S Phase and Is Required for Chromosomal DNA Replication

doi: 10.1091/mbc.E03-02-0088

Figure Lengend Snippet: Dpb2 fails to associate with origin DNA in cdc10-129 cells at 36°C. (A) Exponentially growing 972 cells (lane 1), G2 arrested GD254 cells (lane 2), or G1 arrested GD261 cells (lanes 3 and 4) were subjected to ChIP analysis for the presence of chromatin bound Dpb2. Either magnetic beads conjugated to anti-FLAG antibodies (lanes 1, 2 and 4) or magnetic beads alone (lane 3) were used for immunoprecipitation. The relative ratio of immunoprecipitated DNA to total DNA was plotted. No enrichment of Dpb2 binding to ars2004 is observed in cdc10-129 cells arrested in G1 (lane 4). The levels of binding to either non-ARS or ARS-associated chromatin in cdc10 cells is equivalent to the level detected in either G2 arrested (lane 2) or exponentially growing cells (lane 1). Very little chromatin is precipitated from cdc10 cells in the absence of antibodies (lane 3). (B) ChIP analysis using anti-FLAG antibodies for detection of Pol ε-Cterm at ars2004.

Article Snippet: The lysate was then clarified by centrifugation (12,000 g , 15 min) at 4°C, and 90% of the supernatant was incubated with anti-FLAG or anti-Mcm6p antibodies bound to magnetic beads (Dynal Biotech, Oslo, Norway).

Techniques: Magnetic Beads, Immunoprecipitation, Binding Assay