Enzyme assay experiment
Summary
In this unit, a simpler method was used to determine its stimulatory effect on core RNA polymerase transcription from a non-specific template poly(dAT) start. This experiment was derived from the Laboratory Guide for Protein Purification and Characterization by Houzhu Zhu.
Operation method
Enzyme assay experiment
Materials and Instruments
Core RNA Polymerase Pure σ32 Standard Purified σ32 Sample Core RNA Polymerase-σ32 Complex Sample [α-32P]UTP Termination Solution DEAE-Cellulose (DE81) Paper P04 Wash Buffer Ethanol Reaction Solution
Water Bath
Move
Materials and equipment
Core RNA polymerase (2.3 mg/ml)
Pure σ32 Standard (0.8 mg/ml)
Purified σ32 sample, eluted from a POROS 50S cation exchange column
Core RNA polymerase-σ32 complex sample, eluted from an immunoaffinity column
[ α-32P ]UTP (with 2.5uCi/ml reaction solution)
Water bath, set at 37°C
Termination solution (0.2mol/LEDTA)
DEAE-cellulose (DE81) paper sheets
P04 Wash Buffer (5% Na2 HP04)
Ethanol (95%)
Reagents
Reaction solution
(For formulations, see "Preparation of Reagents", PP.184~189)
Shoving Procedure
1) Take a 50-ul aliquot of reaction solution (containing 2.5uCi[ α-32P ]UTP/ml) by pipette and add it to each tube on ice.
2) Add 5ul of appropriately diluted σ32 sample to each tube and mix well. One sample should be the standard preparation and the other sample should be the core RNA polymerase-σ32 complex eluted from the immunoaffinity column. Also ensure that there is a "no σ32 " blank to determine the incorporation of only core RNA polymerase and an "unincubated" blank (tube on ice with termination solution, but not incubated at 37°C) to determine background.
3) Add 5ul of diluted core RNA polymerase (1ug total) to each tube except the tube containing the core RNA polymerase-σ32 complex and one tube containing pure σ32 standard.
4) Incubate each tube at 37°C for 10 min, then add 10ul of termination solution to terminate the reaction.
5) Take 25ul of reaction solution from each tube and spot it on a DEAE-cellulose paper plate.
6) Place the paper sheet in a beaker and wash four times with pre-cooled P04 wash buffer, 100 ml each time for 5 min. the first two washes were poured into the radioactive waste container. The first two washes were poured into the radioactive waste container. Finally, the paper was washed once with H20 only and once with 95% ethanol.
7) Dry the paper and count the radioactivity.
8) For each σ32 sample, take the spiked amount (minus the background of the "unincubated" blank) and plot it against the spiked amount. Determine the activity of each sample relative to a known σ32 standard. The relative activity can be calculated from the protein concentration of each sample.
Note: Another activity is its ability to bind to the core RNA polymerase. This can be determined by analyzing the conversion of Core RNA Polymerase to the Core RNA Polymerase-σ32 complex by non-denaturing gel electrophoresis, as described in Experiment 5 of this module (PP.169-171).
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