Inclusion body precipitation dissolution assay

Summary

This experiment describes the amount of sodium N-dodecyl sarcosinate required for solubilizing inclusion body precipitation. This experiment is from the Laboratory Guide for Protein Purification and Identification, by Zhu Houzhu.

Operation method

Inclusion body precipitation dissolution assay

Materials and Instruments

Buffer A Sodium N-dodecyl sarcosinate (SKL) Coomassie blue (CBB) Staining solution Decolorizing solution Reagents for rapid protein spot blotting test
SDS-PAGE Electrophoresis Unit Polyacrylamide Small Gel (10%) Molecular Weight Reference Standard

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Materials and equipment

SDS-PAGE electrophoresis device

Polyacrylamide small gel (10%)

Molecular weight standard reference

Reagents

Buffer A

Sodium N-dodecyl sarcosinate (SKL) (20% aqueous stock solution)

Caulmers Brilliant Blue (CBB) Staining Solution

Decolorizing solution

Reagents for rapid protein blotting (see P.172)

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

Operating procedure

1) For the 2nd 2% D0C suspension (see p.149), take 6x50-ul aliquots before centrifugation. These samples are then centrifuged in a microcentrifuge at full speed for 1 min and the supernatant discarded.

2) To each precipitate, add 100 ul of Buffer A containing 0%, 0.1%, 0.2%, 0.3%, and 0.6% SKL, respectively. Resuspend the precipitates vigorously for 10 min, then centrifuge for 1 min in a microcentrifuge, and remove the supernatant for a rapid protein blotting assay (see pp. 172-173) to observe the amount of precipitate.

3) Perform SDS gel electrophoresis of each supernatant, and perform rapid protein spot blotting test with samples A and B.

4) For gel electrophoresis analysis, on a 10% SDS gel, add 20ul of each of samples A, B, C, D and 0.1% SKL supernatant, 0.2% SKL supernatant, 0.3% SKL supernatant, 0.4% SKL supernatant, 0.6% SKL supernatant, 3ul of molecular weight standard reference, and electrophoresis for about 60 min at 120V (20mA).

5) The gel was stained with CBB staining solution for 30 min, then rinsed and decolorized in decolorizing solution until the bands appeared.

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