Primary Rat hippocampal and mouse cortical cultures, and BL21-CodonPlus (De3)-RIL competent cells (Agilent Technologies, cat. # 230280).
Bl21 codonplus de3 ril competent cells
Manufactured by Agilent Technologies
Sourced in United States
BL21-CodonPlus (DE3)-RIL competent cells are a strain of Escherichia coli that are designed for the high-level expression of recombinant proteins. These cells contain rare tRNA genes to enhance the expression of proteins from organisms with codon biases that differ from E. coli.
Automatically generated - may contain errors
Lab products found in correlation
Phusion polymerase, by New England Biolabs (4 mentions)
Prescission protease, by GE Healthcare (3 mentions)
Sp sepharose, by GE Healthcare (2 mentions)
Hiload 16 60 superdex 200 pg gel filtration column, by GE Healthcare (2 mentions)
Superdex 200 10 300 gl column, by GE Healthcare (1 mentions)
Histrap, by GE Healthcare (1 mentions)
Microfluidizer, by Microfluidics (1 mentions)
Neb 5 alpha competent e coli high efficiency, by New England Biolabs (1 mentions)
His gravitrap, by GE Healthcare (1 mentions)
Gravitrap, by Cytiva (1 mentions)
Ni nta agarose, by Qiagen (1 mentions)
Protein labeling kit red nhs 2nd generation, by NanoTemper (1 mentions)
Monolith nt 115, by NanoTemper (1 mentions)
Cas9 plasmid, by Addgene (1 mentions)
Imperial protein stain, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
21 protocols using bl21 codonplus de3 ril competent cells
1
Culturing Rat Hippocampal and Mouse Cortical Cells
2
Purification of D8 Protein from E. coli
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BL21-CodonPlus(DE3)-RIL competent cells (Agilent) were transformed with one of the D8 expression vectors and grown in LB media with 1 mM Ampicillin at 37°C until OD600 ∼0.6. Protein expression was then induced with 1 mM IPTG for 4 hrs at 37°C, while shaking at 230 rpm. Cells were pelleted and resuspended in lysis buffer containing 100 mM Tris pH 8.0, 300 mM NaCl, 0.5 mM EDTA, 20 mM Imidazole, 0.2 mM PMSF and lysed under 20000 psi pressure using a microfluidizer (Microfluidics). Cell lysate was clarified at 50,000 g for 20 min. Supernatant was loaded onto 5 mL Ni-NTA column (His-Trap, GE). Bound D8 protein was eluted with 20 mM Tris pH 8.0, 300 mM NaCl, 200 mM Imidazole. After overnight dialysis against 20 mM Tris pH 8.0, 200 mM NaCl, the sample was concentrated and subjected to SEC using a Superdex 200 10/300GL column (GE) in the same buffer. The monomeric peak with VE≅16.5 mL and oligomeric peak with VE≅11.7 mL were collected in separate fractions.
3
Bacterial Cell Culture for Protein Expression
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NEB 5-alpha Competent E. coli (High Efficiency) (New England Biolabs) and BL21-CodonPlus (DE3)-RIL Competent Cells (Agilent technologies) were grown at 37°C, unless otherwise stated, in LB media (1% bactotryptone, 1% yeast extract and 0.5% sodium chloride) containing 75 μg/mL ampicillin.
4
Engineered RANKL Mutations Disrupt RANK Binding
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Example 1
RANKL residues forming salt bridges or hydrogen bonds with RANK were targeted for site directed mutagenesis using the program PISA (European Bioinformatics Institute, Cambridgeshire, UK) and the RANK/RANKL co-crystal structure (9). Loops at the RANK/RANKL interface were disrupted by amino acid insertion. Mutations were introduced into the expression construct, pGEX-GST-RANKL, by PCR using Phusion polymerase (New England BioLabs, Ipswich, Mass.). After verification by nucleic acid sequencing, the mutant RANKL-encoding constructs were transformed into E. coli strain BL21-CodonPlus (DE3)-RIL competent cells (Agilent Technologies Inc., Santa Clara, Calif.) for protein production. Correctly-folded soluble protein was purified from cell lysate on glutathione sepharose (8). The mutant RANKL protein was released from the GST affinity tag by digestion with PreScission™ protease (GE Life Sciences, Piscataway, N.J.).
5
Purification of HIF-2α-ARNT Complex
6
Purification and Antibody Generation for F341_RBP
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The F341_RBP was expressed and purified as previously described using E. coli BL21-CodonPlus (DE3)-RIL competent cells (Agilent Technologies) and 0.5 mM IPTG (isopropyl β-D-1-thiogalactopyranoside) induction (Sørensen et al., 2021a (link)). The F341_RBP was purified using Ni2+-NTA-affinity chromatography (His GraviTrap, GE Healthcare) according to the manufacturer’s instructions and exchanged into PBS buffer using 10 kDA Amicon® Ultra-15 Centrifugal filter units. The purified protein was used to raise polyclonal rabbit antibodies generated by Davids Biotechnologie GmbH (Germany). Anti-F341_RBP serum (anti-RBP serum) was purified on a protein A column and stored at −20°C until use. The binding ability of purified anti-RBP serum to phage F341 was confirmed by standard Western blotting.
7
RANKL Mutational Analysis for RANK Binding
8
Recombinant Enterovirus 71 VP1 Purification
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Recombinant Enterovirus 71 virus particle 1 (rVP1, EU703814.1) was expressed and purified from E. coli, as previously described.28 (link) rVP1 was cloned in the pET28b vector and transformed into BL21-CodonPlus (DE3)-RIL competent cells (Agilent Technologies, USA) for protein expression. rVP1 was expressed in LB medium with 250 μM IPTG, purified using Ni-NTA agarose (Qiagen, Germany) according to the manufacturer’s instructions, and dialyzed with phosphate-buffered saline (PBS). The size of rVP1 was confirmed using SDS-polyacrylamide gel electrophoresis (PAGE).
9
Purification of HIF-2α-ARNT Complex
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To obtain HIF-2α-ARNT complex proteins, the pSJ2-HIF-2α plasmid was co-transformed along with pMKH-ARNT into BL21-CodonPlus (DE3)-RIL competent cells (Agilent Technologies). Following 0.1 mM IPTG induced protein expression overnight at 16 °C, cell pellets were lysed by sonication, and supernatants were applied onto pre-packed His∙Bind resin (Novagen). The bound proteins were further purified using SP Sepharose (GE Healthcare), and the eluted fractions were then loaded on a HiLoad 16/60 Superdex 200pg gel filtration column (GE Healthcare) equilibrated in 20 mM Tris (pH 8.0) and 400 mM NaCl. DTT was added to the pooled protein peak fractions at 10 mM. The heterodimeric proteins of HIF-2α and ARNT-GFP were prepared similarly as described above, except that the pMKH-ARNT-GFP plasmid was used in the place of pMKH-ARNT. The ARNT-GFP protein was co-expressed and purified in complex with HIF-1α and NPAS3 (plasmids made in our previous studies3 (link),8 (link)), respectively. The single PAS-B domain of HIF-2α was produced by transformation of pSJ2-HIF-2α (241–361) into BL21-CodonPlus (DE3)-RIL, followed by overnight expression, and purification using His-tag affinity chromatography and gel filtration chromatography.
10
Thermophoretic Characterization of RORγt Binding
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For MST screening, we used Monolith NT.115 instrument from Nanotemper to assess the compounds/RORγt interaction. We procured recombinant human RORγt from WuXi and labeled it with Protein Labeling Kit RED-NHS 2nd Generation from Nanotemper (Cat #MO-L011). RORγt was produced by recombinant microbial expression from E. coli cells (BL21-CodonPlus (DE3)-RIL Competent Cells, Cat #230245 from AGILENT). We dissolved RORγt in PBS buffer (pH 7.4) with 0.1% bovine serum albumin (BSA) and 0.05% Tween 20. We kept the concentration of the fluorescently labeled RORγt constant at 25 nM. We added a volume of 5 μL of the corresponding samples in MST capillaries with a final DMSO concentration of 2%. Subsequently, we incubated the samples within the capillaries for 20 min at room temperature prior to the measurements. We detected changes in thermophoretic properties as a change in fluorescence intensity upon incubation of various concentrations of the tested compounds with fluorescently labeled RORγt. We plotted the thermophoresis signal against the compound concentration to obtain a dose–response curves, from which KD values can be deduced. MST data are represented as normalized change in fluorescence (Fnorm) upon ligand binding. Fnorm is the ratio of the fluorescence measured before and during thermophoresis.
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