All PfHAD1 (PlasmoDB ID PF3D7_1033400) alleles were amplified from P. falciparum genomic DNA using the following primers: 5′-CTCACCACCACCACCACCATATGCACGAAATTGTAGATAAGAA-3′ and 5′-ATCCTATCTTACTCACTTATATGTCACAGAATGTCTTCA-3′. The PCR product was cloned by ligation independent cloning into vector BG186149 (link), which introduces an N-terminal 6xHis tag and the construct was verified by Sanger sequencing. BG1861:6xHis-PfHAD1 was transformed into BL21(DE3)pLysS Escherichia coli cells (Life Technologies). Following induction with IPTG, cells were harvested by centrifugation and stored at −20 °C. Pellets were resuspended in lysis buffer containing 10 mM Tris HCl (pH 7.5), 20 mM imidazole, 1 mM MgCl2, 1 mM dithiothreitol (DTT), 1 mg/ml lysozyme, 100 U benzonase, and Complete Mini EDTA-free protease inhibitor tablets (Roche Applied Science). 6xHis-PfHAD1 was bound to nickel agarose beads (Gold Biotechnology), eluted in 300 mM imidazole, 20 mM Tris HCl (pH 7.5), and 150 mM NaCl, and dialyzed in buffer lacking imidazole prior to analysis. Protein was flash frozen and stored at −80 °C.
Escherichia coli bl21 de3 plyss cells
Manufactured by Thermo Fisher Scientific
Sourced in United States
Escherichia coli BL21(DE3)pLysS cells are a common bacterial strain used in molecular biology and protein expression applications. These cells are designed for the inducible expression of recombinant proteins under the control of the T7 promoter system. The pLysS plasmid provides a low level of T7 lysozyme, which helps to suppress basal expression of the target protein prior to induction.
Automatically generated - may contain errors
Lab products found in correlation
Nickel agarose beads, by GoldBio (2 mentions)
Complete mini edta free protease inhibitor tablet, by Roche (2 mentions)
Ni nta agarose, by Qiagen (1 mentions)
Mono s 5 50 gl column, by GE Healthcare (1 mentions)
Nupage 4 12 bis tris protein gel, by Thermo Fisher Scientific (1 mentions)
Superdex 200 10 300 gl column, by GE Healthcare (1 mentions)
Quikchange 2 site directed mutagenesis kit, by Agilent Technologies (1 mentions)
Pet23a plasmid, by GenScript (1 mentions)
7 protocols using escherichia coli bl21 de3 plyss cells
1
Recombinant Expression of PfHAD1 Protein
2
Recombinant Expression of PfHAD1 Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All PfHAD1 (PlasmoDB ID PF3D7_1033400) alleles were amplified from P. falciparum genomic DNA using the following primers: 5′-CTCACCACCACCACCACCATATGCACGAAATTGTAGATAAGAA-3′ and 5′-ATCCTATCTTACTCACTTATATGTCACAGAATGTCTTCA-3′. The PCR product was cloned by ligation independent cloning into vector BG186149 (link), which introduces an N-terminal 6xHis tag and the construct was verified by Sanger sequencing. BG1861:6xHis-PfHAD1 was transformed into BL21(DE3)pLysS Escherichia coli cells (Life Technologies). Following induction with IPTG, cells were harvested by centrifugation and stored at −20 °C. Pellets were resuspended in lysis buffer containing 10 mM Tris HCl (pH 7.5), 20 mM imidazole, 1 mM MgCl2, 1 mM dithiothreitol (DTT), 1 mg/ml lysozyme, 100 U benzonase, and Complete Mini EDTA-free protease inhibitor tablets (Roche Applied Science). 6xHis-PfHAD1 was bound to nickel agarose beads (Gold Biotechnology), eluted in 300 mM imidazole, 20 mM Tris HCl (pH 7.5), and 150 mM NaCl, and dialyzed in buffer lacking imidazole prior to analysis. Protein was flash frozen and stored at −80 °C.
3
Purification and Characterization of Recombinant αA-Crystallin
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Recombinant wild-type αA-crystallin (αAWT) and αAG98R-crystallin were expressed in Escherichia coli BL21(DE3)pLysS cells (Invitrogen, Carlsbad, CA) and purified using column chromatography, as described previously [16 (link)]. The purity of the protein was checked with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE), and the molecular mass was determined with mass spectrometry (MS). The minichaperone peptide D71FVIFLDVKHFSPEDLTVK88 was supplied by GenScript (Piscataway, NJ). Biotinyl-D71FVIFLDVKH(Bpa)SPEDLTVK88 was obtained from Apptec (Louisville, KY). The peptides used in the study were >95% pure as determined with high-performance liquid chromatography (HPLC) and mass spectrometry. All buffers for experimental measurements were made from American Chemical Society–grade reagents.
4
Expression and Purification of Recombinant Antigens
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Expression and purification of the His-tagged recombinant antigens Lci10 and Lci13 was performed as described (23 (link)), using Escherichia coli BL21(DE3) pLysS cells (Invitrogen®, Carlsbad, CA, USA) transformed with pRSET-derived plasmids containing the Lci10 or Lci13 genes. Nickel-nitrilotriacetic acid (Ni-NTA) Agarose (QIAGEN®, Germantown, MD, USA) was used for purification. Protein concentrations were determined by the Bradford’s method (34 (link)). SDS-PAGE gels (15%) were performed to check the effectiveness of each purification.
5
Recombinant AlboD7L1 Protein Production
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Synthetic AlboD7L1 gene was subcloned into pET-17b (Bio Basic Inc., Markham, ON, Canada) for expression in Escherichia coli BL21 (DE3) pLysS cells (Invitrogen, San Diego, CA, USA). Protein expression was carried out as previously described [33 (link)]. Inclusion bodies were refolded using 300 mM arginine, 50 mM Tris, pH 8.0. AlboD7L1 recombinant protein was purified, first by size exclusion chromatography on a HiPrep 16/60, followed by cation exchange chromatography using Mono S 5/50 GL column (GE Healthcare Life Science, Piscataway, NJ, USA). A final analytical size exclusion chromatography was performed using a Superdex 200 10/300 GL column (GE Healthcare Life Science, Piscataway, NJ, USA). AlboD7L1 purified protein was separated in NuPAGE 4–12% Bis-Tris Protein Gels (Life Technologies, Carlsbad, CA, USA) and visualized by Coomassie stain. Protein identity was confirmed by Edman degradation at the Research Technologies Branch, NIAID, NIH.
6
Overexpression of βarr2 in E. coli
7
Recombinant Mutant αB-Crystallin Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Gene-expressing human αB∆21–28, ∆54–61 (without the C-terminal His-tag) and cloned onto pET23a (+) plasmid was obtained from GenScript USA Inc., Piscataway, NJ, USA. The in-frame T7 tag was removed by PCR using QuikChange II Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA, USA). The reading frame and the mutations were confirmed by DNA sequencing. The Plasmid DNA was transformed into Escherichia coli BL21(DE3) pLysS cells (Invitrogen Corp., Carlsbad, CA, USA) for expressing the mutant protein.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!