Methods and kits for isolation and analysis of a chromatin region

摘要

The present invention encompasses methods of identifying proteins and protein modifications of proteins specifically associated with a chromatin.

主权项

1. A method of identifying proteins, including proteins comprising posttranslational modifications, specifically associated with a target chromatin in a cell, the method comprising:
a) providing:
i) a first cell sample comprising nucleic acid binding proteins and the target chromatin, wherein the target chromatin is tagged by contacting the target chromatin with a tag capable of specifically recognizing and binding one or more portions of the target chromatin and wherein the tag comprises a nucleic acid engineered to have binding specificity for a nucleic acid sequence component of the target chromatin, a protein that associates with the nucleic acid engineered to have binding specificity for a nucleic acid sequence component of the target chromatin, and an affinity handle, andii) a second cell sample comprising nucleic acid binding proteins and the target chromatin, wherein the target chromatin is not tagged by contacting the target chromatin with a non-functional tag that is not capable of specifically recognizing and binding one or more portions of the target chromatin and wherein the non-functional tag comprises a protein capable of associating with a nucleic acid engineered to have binding specificity for a nucleic acid sequence component of the target chromatin and an affinity handle, wherein the non-functional tag does not comprise a nucleic acid engineered to have binding specificity for a nucleic acid sequence component of the tartget chromatin,wherein the first cell sample and the second cell sample are lysed; b) performing affinity purification on each lysed cell sample in (a) using a substrate capable of binding the affinity handle, wherein affinity purification of the first cell sample results in isolation of affinity handle bound to tagged target chromatin bound to nucleic acid binding proteins and affinity purification of the second cell sample results in isolation of nucleic acid binding proteins non-specifically bound to the substrate, wherein isolating the affinity handle isolates proteins associated with the tagged target chromatin; c) identifying bound proteins from (b); and d) determining the amount of each bound protein in each cell sample from (b), wherein bound proteins that are enriched in the first cell sample as compared to the second cell sample are specifically associated with the tagged chromatin in the first cell sample.