This experiment describes the amount of polyethyleneimine needed to precipitate σ32 and RNA polymerase. This experiment was derived from Protein Purification and Identification Laboratory Guide by Houzhu Zhu.

Operation method

Precipitation σ32 and RNA polymerase assays

Materials and Instruments

Polyethyleneimine
SDS-PAGE electrophoresis device Polyacrylamide small gels

Move

Materials and equipment

SDS-PAGE electrophoresis device

Polyacrylamide mini-gel (10%)

Reagents

Polyethyleneimine (PEI) (6% reservoir, pH 7.9)

(For the formula, see "Preparation of reagents", pp.184~189)

Operating procedure

1) For 0.2% DOC supernatant (see p.153), take 6x50-ul aliquots. Add 0, 1, 2, 3, 4 and 5 ul of 6% PEI reservoir solution to each sample (so that the PEI concentration is 0%, 0.12%, 0.24%, 0.36%, 0.48%, and 0.6%, respectively), mix thoroughly, and incubate for 5~10 min.

2) Centrifuge 1 ml with a microcentrifuge to separate the precipitate. If necessary, take the supernatant sample for rapid protein spot blotting (see PP.172?173) or SDS gel electrophoresis. Spot blotting should be done immediately.

Results

A typical SDS gel photograph showing the results of a PEI titration experiment is shown in Figure 3-5.

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