A-B Toxin

Modified Protocol

Buffers

Lysis buffer: 50 mM Tris–HCl pH 8.0,1 mM MgCl2,0.4 mg/ml DNase I, 0.4 mg/ml RNase A,1 mg/ml lysozyme.
Wash buffer 1: 20 mM Tris–HCl pH 8.0, 23% (w/v) sucrose,0.5 %(v/v) Triton X-100, 1 mM EDTA.
Wash buffer 2: 20 mM Tris–HCl pH 8.0, 1 mM EDTA.
Solubilization buffer: 6 M Guanidine HCl(we did not have it so we use 8M urea), 50 mM Tris–HClpH 8.0, 1 mM DTT.

Protocol

1. The steps before harvest the bacteria is the same as before
   

https://2014.igem.org/Team:SUSTC-Shenzhen/Notebook/A-B_Toxin/Purify#2014.8.25(steps before the step9 will be repeated by this protocol)

1. Suspend the pellet in 30 ml of lysis buffer, incubate it at 37°C

for 10–15 min, and sonicate it with a large sonicator tip.

1. Harvest inclusion bodies by centrifuge (25,000 × g for

30 min).

1. Wash inclusion bodies with wash buffer 1 by sonication, cen-

trifuge at 25,000 × g for 15 min and discard the supernatant. Repeat this step for five times.

1. Wash inclusion bodies with wash buffer 2 by sonication,

centrifuge and discard the supernatant.

1. Resuspend inclusion bodies in solubilization buffer, stir at

room temperature for 1–2 h.

1. If the gel result is ideal enough, we can ignore the steps of purification of Ni Column, which will decrease the loss of protein in the purify processes.

Referrence

Protein Expression and Purification of IntegrinI Domains and IgSF Ligands for Crystallography Hongmin Zhang and Jia-huai Wang