Lab Resources

Lab Protocols: Steady State Polymerase Activity Assay (NS5B example)

Rate of GMP Incorporation into a C40 Template without a Primer

Introduction: This protocol describes a method to qualitatively measure the rate of incorporation of the ribonucleotide GMP onto a template of C40 by a polymerase. Data is collected by spotting quenched sample on chromatography paper and exposing it to a phosphor screen. The results are compared to a standard curve of known GTP concentrations and the GTP is spiked with radioactive GTP to reduce the amount of radioactivity needed in the reaction.

Final Assay Conditions:

20 mM HEPES pH 7.3
7.5 mM MnCl2
0.1 mg/mL BSA
2 U/mL RNase Inhibitor
5 mM DTT
20 µM GTP (1000:1 of GTP:GTP[α-32P])
5 µM C40 in terms of C (Template)
0.4 µM Polymerase
100 µL Total volume, 25°C

Protocol:

Make a 1000:1 mixture of GTP and GTP (α-32P). (Final assay conditions should be 20µM GTP : 20 nM GTP[α-32P].)

Add all but GTP into an Eppendorf tube and incubate for 5 minutes at 25°C.

Add the GTP (1000:1 of GTP:GTP[α-32P]) at start.

Remove 10 mL from mix at intervals of 2, 4, 8, 16, 24, 40, and 60 minutes and quench by adding to Eppendorf tubes containing 10 µL of 50 mM EDTA.

Spot 2 µL of the quenched samples from each time point on Whatman DE81 (DEAE) sheet. Be sure to place plastic wrap under the chromatography paper to prevent the sample from soaking into any underlying material.

Wash the sheet 4 times with 250 mL 5% NaH2PO4, pH 7, and dry with air dryer. Wrap the dried chromatography paper in plastic wrap to prevent any contamination of the phorphorscreen with radioactivity during the exposure.

Blank the phosphorscreen on the Image Eraser and expose the spotted chromatography paper in a cassette 30 minutes to 24 hours depending on the amount of GTP [α-32P] used. (20 µM 1000:1 of GTP:GTP [α-32P] can be left over night.)

Analyze the screen with the STORM.

Standard:

The standard curve should be set up with concentrations of GTP from 800 nM to 25 nM GTP using a 1:2 series dilution scheme. For example, the 800nM solution will be 800 nM GTP and 0.8nM GTP[α-32P] and it can be diluted 1:2 to yield 400nM and so on.

Spot 2µl of each dilution on a Whatman DE81 (DEAE) sheet and let dry. Place plastic wrap under the chromatography paper. Do not wash with 5% NaH2PO4.

Wrap this sheet in plastic wrap and expose on a phosphorscreen along with the samples.

Contributed by Richard Allen

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