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Pgex4t3

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The PGEX4T3 is a plasmid vector designed for the expression of recombinant proteins in Escherichia coli. It features a tac promoter for inducible expression, a glutathione S-transferase (GST) gene for affinity purification, and a multiple cloning site for insertion of the target gene.

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Lab products found in correlation

Pgadt7, by Thermo Fisher Scientific (2 mentions) Pureyield plasmid midiprep system, by Promega (1 mentions) Pegfp n1, by Thermo Fisher Scientific (1 mentions) Nanodrop 2000, by Thermo Fisher Scientific (1 mentions) Pcr2.1 topo, by Thermo Fisher Scientific (1 mentions) Anti ha peroxidase antibody, by Roche (1 mentions) Petduet 1, by Thermo Fisher Scientific (1 mentions) Pgbkt7, by Thermo Fisher Scientific (1 mentions) Pcrtm2.1 topo, by Thermo Fisher Scientific (1 mentions)

3 protocols using pgex4t3

1

Cloning and Expression of CiTIA1

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The CiTIA1 ORF was amplified by RT-PCR from the total RNA extracted from CIK cells using the primer pair (F-5ʹ ccgCTCGAGATGATGGACGACGAGCAGC ʹ3 and R-5ʹ cccAAGCTTGTGTGTGTGGTATCCTGCAGT 3ʹ). The amplified fragment was cloned into the eukaryotic expression vector pEGFP-N1 (Invitrogen) and prokaryotic expression vector pGEX-4T-3 (Invitrogen) to generate pEGFP-TIA1 and pGST-TIA1, respectively. Plasmid extraction and purification was performed using the PureYield Plasmid Midiprep System (Promega). The quantity and quality of the extracted DNA was determined by Nanodrop 2000 (Thermo). The plasmid DNA was stored at −20 °C.
Song L., Wang H., Wang T, & Lu L. (2015). Sequestration of RNA by grass carp Ctenopharyngodon idella TIA1 is associated with its positive role in facilitating grass carp reovirus infection. Fish & Shellfish Immunology, 46(2), 442-448.
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2

Plasmid and Antibody Acquisition Protocol

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pCR2.1-TOPO, pETDuet-1, pGEX4T3, and pGADT7 plasmids were purchased from Invitrogen, Novagen, Life Sciences, and Clontech respectively. pCAM-BSD and pCAM-BSD-HA plasmids were kind gifts from Dr. C. Doerig (Monash University, Melbourne, Australia).
Monoclonal anti-HA, anti-penta His, anti-GST, anti-H3, and anti-HA peroxidase antibodies were purchased from Roche, Qiagen, Sigma-Aldrich, Millipore, and Abcam respectively. Anti-actin1 and anti-SOD1 antibodies were used as previously described (Daher et al., 2006 (link), 2010 (link)).
Tellier G., Lenne A., Cailliau-Maggio K., Cabezas-Cruz A., Valdés J.J., Martoriati A., Aliouat E.M., Gosset P., Delaire B., Fréville A., Pierrot C, & Khalife J. (2016). Identification of Plasmodium falciparum Translation Initiation eIF2β Subunit: Direct Interaction with Protein Phosphatase Type 1. Frontiers in Microbiology, 7, 777.
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3

Reverse Genetic Studies in Plasmodium Species

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Plasmids pCRTM2.1-TOPO®, pQE30, pGEX4T3, pGADT7, and pGBKT7 were purchased from Invitrogen, Qiagen, Life Sciences and Clontech, respectively. Plasmids used in reverse genetic studies in P. falciparum, pCAM-BSD and pCAM-BSD-HA, were kind gifts from Prof. C. Doerig (Monash University, Melbourne, VIC, Australia). Plasmids used in P. berghei reverse genetic studies, p-TRAD4Ty-TetO7-HA-hDHFR and pBS-DHFR, were given by Prof. D. Soldati-Favre (University of Geneva, Switzerland) and Prof. R. Tewari (University of Nottingham, United Kingdom), respectively.
All primers used in this study are indicated in Supplementary Table 1.
Lenne A., De Witte C., Tellier G., Hollin T., Aliouat E.M., Martoriati A., Cailliau K., Saliou J.M., Khalife J, & Pierrot C. (2018). Characterization of a Protein Phosphatase Type-1 and a Kinase Anchoring Protein in Plasmodium falciparum. Frontiers in Microbiology, 9, 2617.
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