pET30 Ek/LIC-Colα3(IV) NC1

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	Five ml overnight bacterial culture was used to inoculate 500 ml LB supplemented with 100 µg/ml carbenicillin (Life Technologies). Bacteria were allowed to grow until the OD600 reached ~0.65 and 0.1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) was used to induce production of recombinant protein for 3.5 h at 37°C. Bacteria were harvested at 6,000 × g 10 min.	All bacterially expressed Colα3(IV) NC1 was found in the inclusion bodies (insoluble fraction). Bacteria were lysed and inclusion bodies were extracted by BugBuster Plus Lysonase kit (EMD Millipore) according to the manufacturer protocol.

Protocol tips
Five ml overnight bacterial culture was used to inoculate 500 ml LB supplemented with 100 µg/ml carbenicillin (Life Technologies). Bacteria were allowed to grow until the OD600 reached ~0.65 and 0.1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) was used to induce production of recombinant protein for 3.5 h at 37°C. Bacteria were harvested at 6,000 × g 10 min.
Downstream tips
All bacterially expressed Colα3(IV) NC1 was found in the inclusion bodies (insoluble fraction). Bacteria were lysed and inclusion bodies were extracted by BugBuster Plus Lysonase kit (EMD Millipore) according to the manufacturer protocol.

Publication protocol

Five ml overnight bacterial culture was used to inoculate 500 ml LB supplemented with 100 µg/ml carbenicillin (Life Technologies). Bacteria were allowed to grow until the OD600 reached ~0.65 and 0.1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) was used to induce production of recombinant protein for 3.5 h at 37°C. Bacteria were harvested at 6,000 × g 10 min. All bacterially expressed Colα3(IV) NC1 was found in the inclusion bodies (insoluble fraction). Bacteria were lysed and inclusion bodies were extracted by BugBuster Plus Lysonase kit (EMD Millipore) according to the manufacturer protocol. 6 M guanidine HCl, 0.1 M NaH2PO4 and 10 mM TRIS-HCl, pH 8.5 was used to dissolve inclusion bodies. Ni2+-Sepharose high performance resin (GE Healthcare) was used to purify the histidine (His)-tagged Colα3(IV) NC1 domain in a packed column (Bio-rad).

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Manufacturer protocol

Download the product protocol from C. Yan Cheng, The Mary M. Wohlford Laboratory for Male Contracep for pET30 Ek/LIC-Colα3(IV) NC1 below.

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