Protocol for E. coli RNA Polymerase Holoenzyme
Protocol for E. coli RNA Polymerase Holoenzyme
Protocol for E. coli RNA Polymerase Holoenzyme
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E. <strong>coli</strong> <strong>RNA</strong> <strong>Polymerase</strong> <strong>Holoenzyme</strong><br />
Cat. Nos. S90050 and S90250<br />
E. <strong>coli</strong> <strong>RNA</strong> <strong>Polymerase</strong> <strong>Holoenzyme</strong> plays a central role in controlling gene expression<br />
in the bacterium through the synthesis of <strong>RNA</strong> transcripts. Epicentre’s preparation of<br />
<strong>Holoenzyme</strong> is 100% saturated with sigma subunit (σ70) and thus initiates <strong>RNA</strong> synthesis<br />
specifically at promoter sequences on native bacterial or bacteriophage DNA.* A method<br />
to assay enzyme function is presented in Chamberlain et al. 1<br />
The enzyme is supplied at a unit concentration of 1 U/μl (0.50 μg/μl, 2.0 x 10 3 U/mg) and<br />
is purified from the rifampicin-sensitive strain BL21.<br />
Product Specifications<br />
Storage: Store only at –70°C.<br />
Storage Buffer: E. <strong>coli</strong> <strong>RNA</strong> <strong>Polymerase</strong> <strong>Holoenzyme</strong> is supplied in a 50% glycerol<br />
solution containing 50 mM Tris-HCl (pH 7.5), 250 mM NaCl, 0.1 mM EDTA, and 1 mM<br />
dithiothreitol (DTT).<br />
Unit Definition: One unit catalyzes the incorporation of 1 nmol of ribonucleoside<br />
triphosphates (NTPs) into <strong>RNA</strong> in 10 minutes at 37°C.<br />
Quality Control: E. <strong>coli</strong> <strong>RNA</strong> <strong>Polymerase</strong> <strong>Holoenzyme</strong> is function-tested in a reaction<br />
containing: 1X E. <strong>coli</strong> <strong>RNA</strong> <strong>Polymerase</strong> Transcription Buffer, 2 mM DTT, 0.25 mM NTPs, and<br />
1 μg T7 D111 DNA as template. The enzyme activity is assayed in a reaction containing:<br />
1X E. <strong>coli</strong> <strong>RNA</strong> <strong>Polymerase</strong> Transcription Buffer, 10 mM DTT, 0.5 mM NTPs, and 1 μg T7<br />
D111 DNA as template.<br />
5X E. <strong>coli</strong> <strong>RNA</strong> <strong>Polymerase</strong> Transcription Buffer: 0.2 M Tris-HCl (pH 7.5), 0.75 M KCl,<br />
50 mM MgCl 2<br />
, and 0.05% Triton® X-100. Optimum buffer composition, including the<br />
concentration of DTT, will vary based on the experimental application.<br />
Contaminating Activity Assays: E. <strong>coli</strong> <strong>RNA</strong> <strong>Polymerase</strong> <strong>Holoenzyme</strong> is free of<br />
detectable DNA exo- and endonuclease, and RNase activities.<br />
Reference:<br />
1. Chamberlin, M.J. et al., (1979) J. Biol. Chem. 254, 10061.<br />
*<strong>Holoenzyme</strong> refers to E. <strong>coli</strong> <strong>RNA</strong> <strong>Polymerase</strong> with the sigma 70 (σ 70 ) as determined by SDS-PAGE analysis and by initiation of transcription<br />
from the host promoter on bacteriophage T7 DNA. The presence of other minor sigma factors (e.g., σ 35 ), transcription factors (e.g., GreA or<br />
GreB), or transcription accessory proteins are not excluded.<br />
www.epicentre.com Lit. # 014 • 6/2011 1
E. <strong>coli</strong> <strong>RNA</strong> <strong>Polymerase</strong> <strong>Holoenzyme</strong><br />
Related Products: The following products are also available:<br />
– E. <strong>coli</strong> <strong>RNA</strong> <strong>Polymerase</strong> Core Enzyme<br />
– NTP Solutions<br />
– Tagetin <strong>RNA</strong> <strong>Polymerase</strong> Inhibitor<br />
– E. <strong>coli</strong> DNA <strong>Polymerase</strong> I<br />
Tagetin is a trademark of Epicentre, Madison, Wisconsin.<br />
Triton is a registered trademark of Rohm & Haas, Philadelphia, Pennsylvania.<br />
Visit our technical blog: epicentral.blogspot.com<br />
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Notes<br />
techhelp@epicentre.com • (800) 284-8474 3
726 Post Road, Madison, WI 53713 (800) 284-8474 (608) 258-3080 Fax (608) 258-3088<br />
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