Precipitation σ32 and RNA polymerase assays
Precipitation σ32 and RNA polymerase assays
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 Move Materials and equipment For more product details, please visit Aladdin Scientific website.
SDS-PAGE electrophoresis device Polyacrylamide small gels
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. 