This experiment describes the amount of salt needed to elute Σ32 and RNA polymerase from PEI precipitation. This experiment is from the Protein Purification and Identification Lab Guide by Houzhu Zhu.

Operation method

Elution of σ32 from PEI precipitation and RNA polymerase assays

Materials and Instruments

Buffer A Buffer A containing 0, 0.3, 0.5, 0.7, 0.9, and 1.1 mol LNaCl
SDS-PAGE electrophoresis device Polyacrylamide microglue

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Materials and equipment

SDS-PAGE electrophoresis device

Polyacrylamide mini-gel (10%)

Reagents

Buffer A

Buffer A containing 0, 0.3, 0.5, 0.7, 0.9 and 1.1 mol/L NaCl

(For the recipe, see "Preparation of Reagents", PP.184~189)

Operating Procedures

1) PEI suspension (see p.153) Take 6X50ul aliquots before centrifugation. Centrifuge each sample in a microcentrifuge for lmin, and then resuspend the precipitate with 50ul of Buffer A containing 0, 0.3, 0.5, 0.7, 0.9, and 1.1mol/LaCl, respectively.

2) Mix well and leave for 10~15 mi, then centrifuge for lmin in a microcentrifuge, take the supernatant samples and analyze them by rapid protein spot blotting (see pp.l72~173) and SDS-PAGE electrophoresis, if necessary.

Results

Typical SDS gel photographs showing the results of NaCl elution experiments are shown in Figures 3-5.

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