Brdu Immunoprecipitation
Brdu Immunoprecipitation - Related Videos
09:14
Examination of Proteins Bound to Nascent DNA in Mammalian Cells Using BrdU-ChIP-Slot-Western Technique
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Cited by 4 •
2016
In this protocol, we describe a novel BrdU-ChIP-Slot-Western technique to examine proteins and histone modifications associated with newly synthesized or nascent DNA.
03:24
BrdU Immunofluorescence Staining: A Technique to Identify Cells in Different Phases of Cell Cycle
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2023
This video describes the protocol for BrdU immunofluorescence staining. The technique allows the identification of cells in different phases of cell cycle. Addition of DNA dyes and antibody labeling allows the detailed analysis of the fate of the S phase cells at later times.
08:08
Co-Immunoprecipitation and Pull-Down Assays
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2023
Co-immunoprecipitation (CoIP) and pull-down assays are closely related methods to identify stable protein-protein interactions. These methods are related to immunoprecipitation, a method for separating a target protein bound to an antibody from unbound proteins. In CoIP, an antibody-bound protein is itself bound to another protein that does not bind with the antibody, this is followed by a separation process that preserves the protein-protein complex. The difference in pull-down assays is that...
08:39
Chromatin Immunoprecipitation
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2023
Histones are proteins that help organize DNA in eukaryotic nuclei by serving as “scaffolds” around which DNA can be wrapped, forming a complex called “chromatin”. These proteins can be modified through the addition of chemical groups, and these changes affect gene expression. Researchers use a technique called chromatin immunoprecipitation (ChIP) to better understand which DNA regions associate with specific histone modifications or other gene regulatory proteins. Antibodies are used to isolate...
21:24
Methylated DNA Immunoprecipitation
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Cited by 43 •
2009
This video demonstrates the protocol for methylated DNA immunoprecipitation (MeDIP). MeDIP is a two day procedure that selectively extracts methylated DNA fragments from a genomic DNA sample using antibodies with specificity for 5 -methylcytosine (anti-5 mC).
FAQs
Antibody selectivity is the key enrichment step: anti-BrdU antibodies bind DNA fragments containing the incorporated label while unlabeled fragments are not specifically captured. After recovery, the enriched fraction can be examined separately from the original DNA preparation. This selective partitioning makes newly synthesized DNA detectable even within a much larger pool of genomic DNA.
Because cells incorporate BrdU during S phase, the label marks DNA synthesized during that period of the cell cycle. Measuring labeled DNA therefore connects sequence recovery with the timing of replication. This relationship allows experiments to examine when particular genomic regions are copied and to compare replication patterns under different biological conditions.
Fragmentation converts extracted DNA into smaller pieces before antibody capture. The anti-BrdU antibody can then selectively recover labeled fragments, which can be analyzed according to their genomic content. This step supports higher-resolution examination of newly synthesized DNA and helps connect enriched material with features such as replication origins or genome-wide replication patterns.
PCR can examine selected DNA regions, whereas microarray analysis and sequencing provide broader views of labeled DNA across the genome. These readouts support different levels of analysis, from targeted detection to genome-wide mapping. The resulting data can be used to investigate replication origins, replication timing, and patterns of DNA synthesis.
A typical workflow begins with BrdU incorporation into DNA synthesized during S phase, followed by DNA extraction and fragmentation. An anti-BrdU antibody is then used to capture labeled fragments, which are recovered for downstream analysis. PCR, microarray analysis, or sequencing can subsequently identify the genomic distribution of the enriched DNA.
This method is useful when researchers need to connect DNA synthesis with broader biological processes. Applications described for it include studying cell-cycle regulation, genome stability, development, and responses to replication stress. By examining enriched newly synthesized DNA, investigators can assess how replication patterns change across these contexts.