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Qiaexpressionist handbook

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The QIAexpressionist Handbook is a comprehensive reference guide that provides detailed information and protocols for the expression and purification of recombinant proteins using the QIAGEN QIAexpress system. The handbook covers various aspects of the process, including vector design, host cell selection, protein expression, and downstream purification methods.

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Lab products found in correlation

E coli rosetta 2 de3 cells, by Merck Group (1 mentions) Incomplete freund s adjuvant, by Merck Group (1 mentions) Ni nta agarose bead, by Qiagen (1 mentions)

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QIAexpressionist (28 citations)

3 protocols using qiaexpressionist handbook

1

Recombinant Cox19 protein expression

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The coding sequence without the stop codon of Cox19 and Cox19EE was amplified from pRS426-Cox19 or Cox19EE and cloned into pET22b (Novagen) upstream of a hexahistidine sequence using the restriction sites BamHI and XhoI. These plasmids were transformed into E. coli Rosetta 2(DE3) cells (Merck, Billerica, MA). The cells were grown at 37°C to an OD600 of 0.6 before expression was induced by the addition of 1 mM isopropyl-β-d-thiogalactoside (IPTG) for 4 h at 37°C. The native purification of the recombinant proteins was performed as described in protocols 9 and 12 of the QIAexpressionist Handbook (Qiagen, Venlo, Netherlands).
Bode M., Woellhaf M.W., Bohnert M., van der Laan M., Sommer F., Jung M., Zimmermann R., Schroda M, & Herrmann J.M. (2015). Redox-regulated dynamic interplay between Cox19 and the copper-binding protein Cox11 in the intermembrane space of mitochondria facilitates biogenesis of cytochrome c oxidase. Molecular Biology of the Cell, 26(13), 2385-2401.
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2

Lbh Fusion Protein Production and Antibody Generation

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The Histidine-tagged fusion protein corresponding to Lbh ORF was produced
according to the QIAexpressionist handbook (Qiagen) and purified using Ni-NTA
agarose beads (Qiagen). Immunization were performed intraperitonally using 100
μg of protein per mice and per injection mixed in with either complete
(primary) or incomplete Freund’s adjuvant (sigma). Eight weeks old Balbc
mice were obtained from Jackson laboratory. Immunization efficiency was
monitored by ELISA on the fusion protein. Three days after the third boost,
splenocytes were fused to sp2/0 cell line using the PEG technique described in
(Harlow and Lane 1989 ). The isotype
of mAb 2B8 developed is an IgG1 Kappa as determined by the IsoStrip kit (Roche
ref. 11493027001). The monoclonal antibody 3D8 against PACSIN2 has been
characterized elsewhere (Cousin, Gaultier et al.
2000).
Weir E., McLinden G., Alfandari D, & Cousin H. (2020). Trim-Away mediated knock down uncovers a new function for Lbh during gastrulation of Xenopus laevis.. Developmental biology, 470, 74-83.
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3

CRISPR-Cas Transformation and Phage Assays

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Cells were made chemically competent using the RuCl method and transformed by applying a heat-shock as described in the QIA expressionist handbook (QIAGEN). For the experiments with Type I-E CRISPR–Cas, E. coli T7 Express (NEB) was transformed with pWUR400, pWUR797 and pWUR800, pWUR801 or pWUR802 (Supplementary Table S1) and used for plaque assays with phage T4. For the in vivo experiments with Type II-A CRISPR–Cas, E. coli T7 Express was transformed with pWUR805 and pWUR806, pWUR809 or pWUR810 (Supplementary Table S1).
Vlot M., Houkes J., Lochs S.J., Swarts D.C., Zheng P., Kunne T., Mohanraju P., Anders C., Jinek M., van der Oost J., Dickman M.J, & Brouns S.J. (2017). Bacteriophage DNA glucosylation impairs target DNA binding by type I and II but not by type V CRISPR–Cas effector complexes. Nucleic Acids Research, 46(2), 873-885.
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