TY - JOUR

T1 - Purification of Elongating RNA Polymerase II and Other Factors from Yeast Chromatin

AU - Svejstrup, Jesper Q.

AU - Petrakis, Thodoris G.

AU - Fellows, Jane

PY - 2003

Y1 - 2003

N2 - This chapter describes procedures for the identification and purification of tightly chromatin-associated factors with particular emphasis on elongating RNA polymerase II. Identification of tightly chromatin-associated proteins in vitro can be done in small scale by the use of a procedure that has been adapted from the work of Diffley and co-workers, used to show that DNA replication proteins such as MCM, Cdc6, and ORC are bound to chromatin. This procedure can be used for quickly establishing if a protein of interest, partitions to both the soluble and the chromatin-associated fraction in vitro. After it has been established that a protein of interest is found in the salt stable chromatin fraction, the protein can be purified from a larger quantity of cells. The chromatin isolation and purification procedure described is based on at least 10 to 20, typically 50 to 80 liters of yeast culture (1011–1013 cells), and breaking cells by bead-beating. Proteins released from chromatin can be purified conventionally or via affinity chromatography. The purification of elongating RNA polymerase II holoenzyme, conventional chromatography with the procedure given has the advantage of gradually co-concentrating components of the fragile holoenzyme.

AB - This chapter describes procedures for the identification and purification of tightly chromatin-associated factors with particular emphasis on elongating RNA polymerase II. Identification of tightly chromatin-associated proteins in vitro can be done in small scale by the use of a procedure that has been adapted from the work of Diffley and co-workers, used to show that DNA replication proteins such as MCM, Cdc6, and ORC are bound to chromatin. This procedure can be used for quickly establishing if a protein of interest, partitions to both the soluble and the chromatin-associated fraction in vitro. After it has been established that a protein of interest is found in the salt stable chromatin fraction, the protein can be purified from a larger quantity of cells. The chromatin isolation and purification procedure described is based on at least 10 to 20, typically 50 to 80 liters of yeast culture (1011–1013 cells), and breaking cells by bead-beating. Proteins released from chromatin can be purified conventionally or via affinity chromatography. The purification of elongating RNA polymerase II holoenzyme, conventional chromatography with the procedure given has the advantage of gradually co-concentrating components of the fragile holoenzyme.

U2 - 10.1016/S0076-6879(03)71036-2

DO - 10.1016/S0076-6879(03)71036-2

M3 - Journal article

C2 - 14712723

AN - SCOPUS:0347536258

VL - 371

SP - 491

EP - 498

JO - Chemical Tools for Imaging, Manipulating, and Tracking Biological Systems: Diverse Methods Based on Optical Imaging and Fluorescence

JF - Chemical Tools for Imaging, Manipulating, and Tracking Biological Systems: Diverse Methods Based on Optical Imaging and Fluorescence

SN - 0076-6879

ER -