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US 2014/0154674 A1Published application
Methods and kits for isolation and analysis of a chromatin region
AssigneeBoard of Trustees of the University of Arkansas
InventorsAlan J. Tackett, Stephanie Byrum, Sean Taverna
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Abstract
The present invention encompasses methods of identifying proteins and protein modifications of proteins specifically associated with a chromatin.
Claims32
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, and
- ii) a second cell sample comprising nucleic acid binding proteins and the target chromatin, wherein the target chromatin is not tagged,
- wherein the proteins in the first cell sample or the second cell sample are metabolically labeled,
- (b) mixing the first cell sample and the second cell sample from (a) with an affinity handle to generate a cell mixture wherein the affinity handle binds protein such that the affinity handle specifically binds the tagged target chromatin and non-specifically binds nucleic acid binding proteins,
- (c) isolating the affinity handle form the mixture in (b) wherein the affinity handle isolated consists of affinity handle bound to tagged target chromatin and affinity handle bound to nucleic acid binding proteins, wherein isolating the affinity handle isolates proteins associated with the tagged target chromatin,
- (d) identifying isolated proteins from (c),
- (e) determining whether each protein from (c) is metabolically labeled, unlabeled, or a mixture of labeled and unlabeled protein, wherein
- i) if the first cell sample from (a) is not metabolically labeled and a protein associated with the target chromatin is not metabolically labeled, then the protein is specifically associated with the tagged chromatin in the cell, and
- ii) if the first sample from (a) is metabolically labeled and a protein associated with the target chromatin is metabolically labeled, then the protein is specifically associated with the tagged chromatin in the cell.
7 more elements
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, and
- ii) a second cell sample comprising nucleic acid binding proteins and the target chromatin, where in the target chromatin is not tagged,
- (b) mixing each of the first cell sample and the second cell sample from (a) with an affinity handle wherein the affinity handle binds protein such that the affinity handle specifically binds the tagged target chromatin and non-specifically binds nucleic acid binding proteins,
- (c) isolating the affinity handle from each mixture in (b) wherein affinity handle isolated from the first cell mixture consists of affinity handle bound to tagged target chromatin and affinity handle bound to nucleic acid binding proteins and affinity handle isolated from the second cell mixture consists of affinity handle bound to nucleic acid binding proteins, wherein isolating the affinity handle isolates proteins associated with the tagged target chromatin,
- (d) identifying bound proteins from (c),
- (e) determining the amount of each bound protein in each mixture from (c), wherein bound proteins that are enriched in the first cell mixture as compared to the second cell mixture are specifically associated with the tagged chromatin in the first cell sample,
4 more elements
Dependencies are inferred from the claim text.