pIVEX2.4c-SnRK2.1

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	The SnRK2.1 protein was expressed in a cell-free expression system based on an E. coli ribosomal extract (S30 extract). S30 extract was prepared as previously reported [18] with some modifications. Briefly, in order to bypass the exogenous addition of commercial creatine kinase (CK) and T7 RNA polymerase (T7 RNAP) into the cell-free reaction mixtures, two types of S30 extracts (CK-T7 extract and BL21 extract) were prepared. The CK-T7 extract was prepared from the E. coli strain BL21 Star (DE3) (Life Technologies) harboring a CK and T7 RNAP expression vector pETDuet-CK-T7 (Figure 1). CK and T7 RNAP were expressed during the cultivation of E. coli via IPTG induction (1.0 mM) at 0.5 OD600.

Protocol tips

The SnRK2.1 protein was expressed in a cell-free expression system based on an E. coli ribosomal extract (S30 extract). S30 extract was prepared as previously reported [18] with some modifications. Briefly, in order to bypass the exogenous addition of commercial creatine kinase (CK) and T7 RNA polymerase (T7 RNAP) into the cell-free reaction mixtures, two types of S30 extracts (CK-T7 extract and BL21 extract) were prepared. The CK-T7 extract was prepared from the E. coli strain BL21 Star (DE3) (Life Technologies) harboring a CK and T7 RNAP expression vector pETDuet-CK-T7 (Figure 1). CK and T7 RNAP were expressed during the cultivation of E. coli via IPTG induction (1.0 mM) at 0.5 OD600.

Publication protocol

The SnRK2.1 protein was expressed in a cell-free expression system based on an E. coli ribosomal extract (S30 extract). S30 extract was prepared as previously reported [18] with some modifications. Briefly, in order to bypass the exogenous addition of commercial creatine kinase (CK) and T7 RNA polymerase (T7 RNAP) into the cell-free reaction mixtures, two types of S30 extracts (CK-T7 extract and BL21 extract) were prepared. The CK-T7 extract was prepared from the E. coli strain BL21 Star (DE3) (Life Technologies) harboring a CK and T7 RNAP expression vector pETDuet-CK-T7 (Figure 1). CK and T7 RNAP were expressed during the cultivation of E. coli via IPTG induction (1.0 mM) at 0.5 OD600. The cells were harvested 2 h after induction and used for preparing the CK-T7 extract. The standard BL21 S30 extract was prepared from the E. coli strain BL21 Star (DE3) as previously reported [18].

Full paper   Login or join for free to view the full paper.

Reviews

Discussion

Papers

Check out relevant papers found by Labettor's AI that are relevant for performing Protein Expression Prokaryotic cells - E. coli SnRK2 A. thaliana using pIVEX2.4c-SnRK2.1 from Yi Wan, Microbiology Institute of Shaanxi, Shaanxi Academy of Sc.

Manufacturer protocol

Download the product protocol from Yi Wan, Microbiology Institute of Shaanxi, Shaanxi Academy of Sc for pIVEX2.4c-SnRK2.1 below.

Videos

Check out videos that might be relevant for performing Protein Expression Prokaryotic cells - E. coli SnRK2 A. thaliana using pIVEX2.4c-SnRK2.1 from Yi Wan, Microbiology Institute of Shaanxi, Shaanxi Academy of Sc. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.