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Kansas University Medical Center

3 ARTICLES PUBLISHED IN JoVE

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JoVE Journal

Generation and Purification of Human INO80 Chromatin Remodeling Complexes and Subcomplexes
Lu Chen 1,2, Soon-Keat Ooi 1, Ronald C. Conaway 1,2, Joan W. Conaway 1,2
1Stowers Institute for Medical Research, 2Department of Biochemistry & Molecular Biology, Kansas University Medical Center

This protocol describes a procedure for generating and purifying wild type and mutant versions of the human INO80 chromatin remodeling complex. Epitope tagged versions of INO80 subunits are stably expressed in HEK293 cells, and complete complexes and complexes lacking specific sets of subunits are purified by immunoaffinity chromatography.

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Biology

Biochemical Assays for Analyzing Activities of ATP-dependent Chromatin Remodeling Enzymes
Lu Chen 1,2, Soon-Keat Ooi 1, Joan W. Conaway 1,2, Ronald C. Conaway 1,2
1Stowers Institute for Medical Research, 2Department of Biochemistry & Molecular Biology, Kansas University Medical Center

Here we describe biochemical assays that can be used to characterize ATP-dependent chromatin remodeling enzymes for their abilities to 1) catalyze ATP-dependent nucleosome sliding, 2) engage with nucleosome substrates, and 3) hydrolyze ATP in a nucleosome- or DNA-dependent manner.

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JoVE Journal

Artificial RNA Polymerase II Elongation Complexes for Dissecting Co-transcriptional RNA Processing Events
Melvin Noe Gonzalez 1, Joan W. Conaway 1,2, Ronald C. Conaway 1,2
1Stowers Institute for Medical Research, 2Department of Biochemistry and Molecular Biology, Kansas University Medical Center

Here, we describe the assembly of RNA polymerase II (Pol II) elongation complexes requiring only short synthetic DNA and RNA oligonucleotides and purified Pol II. These complexes are useful for studying mechanisms underlying co-transcriptional processing of transcripts associated with the Pol II elongation complex.

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