Protein Analysis and Complex Purification

The majority of histone-mediated regulation stems from histone modification, including acetylation, methylation, phosphorylation, ubiquitination and sumoylation. Chromatin immunoprecipitation (ChIP) is a powerful assay to identify proteins that read, write or erase these histone marks. In ChIP analysis, protein:protein and protein:DNA complexes are crosslinked, immunoprecipitated using an antibody against the protein of interest and purified. The DNA sequence of interest then is amplified from the immunoprecipitated material using PCR. Alternatively, the immunoprecipitated DNA can be sequenced or analyzed using microarrays to identify target sequences.

Analysis of protein:DNA interactions often reveals important information chromosomal remodeling and transcription. Protein purification is a fundamental step for analyzing individual proteins and protein complexes and identifying interactions with other proteins, DNA or RNA. A variety of protein purification strategies exist to address desired scale, throughput and downstream applications. There are four basic steps of protein purification: 1) cell lysis, 2) protein binding to a matrix, 3) washing and 4) elution. Because purification of native proteins can be challenging, affinity purification tags are often fused to a recombinant protein of interest such that the tag is used to capture or detect the protein.