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Bl21 ai cells

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BL21-AI cells are a strain of Escherichia coli (E. coli) bacteria that have been genetically engineered for the purpose of recombinant protein expression. These cells are designed to produce high levels of target proteins when induced with the addition of L-arabinose.

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Lab products found in correlation

L arabinose, by GoldBio (4 mentions) Superdex 200, by GE Healthcare (3 mentions) Hitrap heparin, by GE Healthcare (2 mentions) Tev cleavable c terminal mbp fusion tag, by Addgene (2 mentions) Hitrap, by Cytiva (2 mentions) Chitin column, by New England Biolabs (2 mentions) 13c labeled glucose, by Cambridge Isotopes (2 mentions) M9 minimal media, by Cambridge Isotopes (2 mentions) 15n labeled ammonium chloride, by Cambridge Isotopes (2 mentions) Gst resin, by GenScript (1 mentions) Oligonucleotide, by Integrated DNA Technologies (1 mentions) Heparin, by GE Healthcare (1 mentions) S200 size exclusion chromatography, by GE Healthcare (1 mentions) Biogel p4 resin, by Bio-Rad (1 mentions) Q sepharose, by GE Healthcare (1 mentions) Protease inhibitor cocktail, by Merck Group (1 mentions) Enzymes for molecular biology, by New England Biolabs (1 mentions) Isopropyl β d thiogalactopyranoside iptg, by Merck Group (1 mentions)

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BL21-AI (41 citations) E. coli BL21-AI cells (10 citations) BL21-AI One Shot Chemically Competent E. coli cells (2 citations)

12 protocols using bl21 ai cells

1

Arabinose-Induced Protein Expression

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BL21-AI cells (Life Technologies) were transformed with the constructs for protein expression overnight at 15°C in Terrific Broth. Induction of expression was carried out by the primary addition of 0.1% (w/v) final concentration L-Arabinose (GoldBio).
Williams J.S., Wojtaszek J.L., Appel D.C., Krahn J., Wallace B.D., Walsh E., Kunkel T.A, & Williams R.S. (2022). Molecular basis for processing of topoisomerase 1-triggered DNA damage by Apn2/APE2. Cell reports, 41(1), 111448.
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2

Purification and Characterization of Apn2 Protein Variants

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The Apn2FL (full length) and Apn2Cat (residues 1–407) were PCR amplified and subcloned into the pET MBP His6 LIC cloning vector (2Cc-T), containing a TEV-cleavable C-terminal MBP fusion tag (Addgene). Apn2Cat(D222N/E59Q) was cloned by site-directed mutagenesis of the Apn2Cat construct. Apn2FL mutants were cloned by site-directed mutagenesis of the Apn2FL construct. The wedge loop deletion construct was made by replacing Apn2 residues W312 through Y323 with two glycine residues. BL21-AI cells (Life Technologies) were transformed with the constructs for protein expression overnight at 15°C in Terrific Broth. Induction of expression was carried out by the primary addition of 0.1% (w/v) final concentration L-Arabinose (GoldBio). Following Amylose affinity chromatography, MBP fusion proteins were diluted in ddH20 and loaded onto 5-mL ion exchange columns for gradient elution (GE Healthcare; HiTrap Heparin). For crystallography, proteins were subjected to overnight TEV protease cleavage for MBP tag removal prior to ion-exchange chromatography. Purest fractions were pooled and purified by Superdex 200 (GE Healthcare) gel filtration for final polishing. Final purity was assessed by SDS-PAGE and fractions pooled and concentrated for subsequent experiments. The yeast PCNA protein was a kind gift from Andrea Kaminski.
Williams J.S., Wojtaszek J.L., Appel D.C., Krahn J., Wallace B.D., Walsh E., Kunkel T.A, & Williams R.S. (2022). Molecular basis for processing of topoisomerase 1-triggered DNA damage by Apn2/APE2. Cell reports, 41(1), 111448.
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3

Purification and Characterization of Apn2 Protein Variants

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The Apn2FL (full length) and Apn2Cat (residues 1–407) were PCR amplified and subcloned into the pET MBP His6 LIC cloning vector (2Cc-T), containing a TEV-cleavable C-terminal MBP fusion tag (Addgene). Apn2Cat(D222N/E59Q) was cloned by site-directed mutagenesis of the Apn2Cat construct. Apn2FL mutants were cloned by site-directed mutagenesis of the Apn2FL construct. The wedge loop deletion construct was made by replacing Apn2 residues W312 through Y323 with two glycine residues. BL21-AI cells (Life Technologies) were transformed with the constructs for protein expression overnight at 15°C in Terrific Broth. Induction of expression was carried out by the primary addition of 0.1% (w/v) final concentration L-Arabinose (GoldBio). Following Amylose affinity chromatography, MBP fusion proteins were diluted in ddH20 and loaded onto 5-mL ion exchange columns for gradient elution (GE Healthcare; HiTrap Heparin). For crystallography, proteins were subjected to overnight TEV protease cleavage for MBP tag removal prior to ion-exchange chromatography. Purest fractions were pooled and purified by Superdex 200 (GE Healthcare) gel filtration for final polishing. Final purity was assessed by SDS-PAGE and fractions pooled and concentrated for subsequent experiments. The yeast PCNA protein was a kind gift from Andrea Kaminski.
Williams J.S., Wojtaszek J.L., Appel D.C., Krahn J., Wallace B.D., Walsh E., Kunkel T.A, & Williams R.S. (2022). Molecular basis for processing of topoisomerase 1-triggered DNA damage by Apn2/APE2. Cell reports, 41(1), 111448.
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4

Arabinose-Induced Protein Expression

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BL21-AI cells (Life Technologies) were transformed with the constructs for protein expression overnight at 15°C in Terrific Broth. Induction of expression was carried out by the primary addition of 0.1% (w/v) final concentration L-Arabinose (GoldBio).
Williams J.S., Wojtaszek J.L., Appel D.C., Krahn J., Wallace B.D., Walsh E., Kunkel T.A, & Williams R.S. (2022). Molecular basis for processing of topoisomerase 1-triggered DNA damage by Apn2/APE2. Cell reports, 41(1), 111448.
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5

Purification of PB2-NLD:Importin α Complexes

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GST-PB2-NLD and untagged or 6xHis tagged importin α isoforms were co-expressed in BL21-AI cells (Invitrogen), induced with 0.1 mM IPTG and 0.1% arabinose, and grown overnight at 20°C. Cell pellets were resuspended in Lysis buffer (20 mM Tris, pH 8.0, 150 mM NaCl, 1 mM EDTA, 5 mM BME, 1 mM PMSF, and 0.2% Tween-20) and lysed by sonication. Clarified lysates were incubated with GST resin (GenScript), and beads were washed with Complex buffer (20mM Tris, pH 8.0, 150 mM NaCl, 1 mM EDTA, 5 mM BME, 0.1 mM PMSF). The PB2- NLD:importin α complexes were removed from immobilized GST-resin by the overnight application of PreScission Protease. The 6xHis tag was removed by the overnight application of TEV protease. MBP-cargos were purified as in (Pumroy et al., 2012 (link)). GST-importin α isoforms were expressed in BL21-AI cells and purified as in (Nardozzi et al., 2010a (link)). Complexes, MBP-cargos and GST-importin α isoforms were further purified by size exclusion chromatography on a Superdex 200 or 75 16/60 (GE Healthcare) in Complex buffer and concentrated by ultrafiltration.
Pumroy R.A., Ke S., Hart D.J., Zachariae U, & Cingolani G. (2015). Molecular determinants for nuclear import of Influenza A PB2 by importin α isoforms 3 and 7. Structure (London, England : 1993), 23(2), 374-384.
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6

Recombinant Protein Expression in BL21-AI

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pDEST17-based plasmids containing proT7-RBS-6xHis-CDS-T7term expression cassettes (proT7: T7 promoter, RBS: ribosome-binding site, 6xHis: polyhistidine tag and T7term: T7 transcription terminator) were transformed into BL21-AI cells (Invitrogen). Cultures were grown in LB to an OD600 of about 0.4 and expression was induced with 0.2% L-arabinose.
Busche M., Acatay C., Martens S., Weisshaar B, & Stracke R. (2021). Functional Characterisation of Banana (Musa spp.) 2-Oxoglutarate-Dependent Dioxygenases Involved in Flavonoid Biosynthesis. Frontiers in Plant Science, 12, 701780.
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7

Affinity Purification of NpR6012g4 Protein

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The protein sample in this study consists of 180 native residues (M583-G762, Fig. 1) after removal of a C-terminal intein-CBD tag used for affinity purification (Kim et al, 2012 (link)). NpR6012g4 was expressed in BL21-AI cells (Invitrogen) grown in M9 minimal media supplemented with ALA (100 μM), 15N-labeled ammonium chloride, and/or 13C-labeled glucose (Cambridge Isotopes) using a published system for induction of protein expression and chromophore biosynthesis (Gambetta & Lagarias, 2001 (link)). Affinity purification of NpR6012g4 using a chitin column (NEB) followed our previous procedure (Kim et al, 2012 (link); Rockwell et al, 2012 (link); Rockwell et al, 2015a (link); Rockwell et al, 2015b (link)). Peak eluted fractions were pooled for overnight dialysis into 10 mM sodium phosphate (pH 7.4) supplemented with 1 mM EDTA to remove residual metal ions followed by final overnight dialysis into 10 mM sodium phosphate (pH 7.4). The protein was concentrated to approximately 0.7 mM, and D2O was added to 7% (v/v). Dark reversion of the metastable green-absorbing state under these conditions was < 10% after 24 hours at 298 K as reported previously (Rockwell et al, 2015b (link)). All subsequent manipulations were performed on samples kept in darkness.
Lim S., Yu Q., Rockwell N.C., Martin S.S., Clark Lagarias J, & Ames J.B. (2015). 1H, 13C, and 15N Chemical Shift Assignments of Cyanobacteriochrome NpR6012g4 in the Green-Absorbing Photoproduct State. Biomolecular NMR assignments, 10(1), 157-161.
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8

Preparation of Labeled Photoreceptor Protein

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The protein sample in this study consists of 180 native residues (M583-G762, Fig. 1) after removal of a C-terminal intein-CBD tag used for affinity purification (Kim et al, 2012 (link)). NpR6012g4 was expressed in BL21-AI cells (Invitrogen) grown in M9 minimal media supplemented with ALA (100 μM), 15N-labeled ammonium chloride, and/or 13C-labeled glucose (Cambridge Isotopes) using a published system for induction of protein expression and chromophore biosynthesis (Gambetta & Lagarias, 2001 (link)). Affinity purification of NpR6012g4 using a chitin column (NEB) followed our previous procedure (Kim et al, 2012 (link); Rockwell et al, 2012 (link); Rockwell et al, 2015a (link); Rockwell et al, 2015b (link)). Peak eluted fractions were pooled for overnight dialysis into 10 mM sodium phosphate (pH 7.4) supplemented with 1 mM EDTA to remove residual metal ions followed by final overnight dialysis into 10 mM sodium phosphate (pH 7.4). The protein was concentrated to approximately 0.7 mM, and D2O was added to 7% (v/v). Dark reversion of the metastable green-absorbing state under these conditions is < 10% after 24 hours at 298 K as reported previously (Rockwell et al, 2015b (link)). All subsequent manipulations were performed in darkness.
Yu Q., Lim S., Rockwell N.C., Martin S.S., Lagarias J.C, & Ames J.B. (2015). 1H, 15N, and 13C chemical shift assignments of cyanobacteriochrome NpR6012g4 in the red-absorbing dark state. Biomolecular NMR assignments, 10(1), 139-142.
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9

Purification of AriAB Protein Complex

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AriA and AriB-strep were co-expressed from a pRSF-Duet plasmid in BL21-AI cells (Invitrogen). Cultures grown at 37°C to 0.4–0.5 OD600 were induced with 0.1 mM IPTG and 0.1% L-arabinose. Following induction, the cultures were incubated overnight at 16°C. Cells were pelleted (3,000 × g, 10 min, at 4°C), resuspended in lysis buffer (25 mM Tris, pH=8.5, 150 mM NaCl, 1 mM EDTA) and lysed by sonication. Cellular debris was removed by centrifugation (10,000 × g, 25 min, at 4°C) and AriAB complex was purified by affinity chromatography on 5 mL streptactin resin (IBA) and eluted with 2.5 mM desthiobiotin (IBA) in lysis buffer. The eluted protein was concentrated (100k MWCO PES Spin-X UF concentrator, Corning) and further purified by size exclusion chromatography (Sup200 column, Cytiva) in lysis buffer with 2% glycerol. Fractions of interest were combined and concentrated to 5μM (100k MWCO PES concentrator, Pierce) and used immediately for vitrification on cryo-EM grids.
Burman N., Belukhina S., Depardieu F., Wilkinson R.A., Skutel M., Santiago-Frangos A., Graham A.B., Livenskyi A., Chechenina A., Morozova N., Zahl T., Henriques W.S., Buyukyoruk M., Rouillon C., Shyrokova L., Kurata T., Hauryliuk V., Severinov K., Groseille J., Thierry A., Koszul R., Tesson F., Bernheim A., Bikard D., Wiedenheft B, & Isaev A. (2024). Viral proteins activate PARIS-mediated tRNA degradation and viral tRNAs rescue infection. bioRxiv.
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10

Fibril Protein Expression in E. coli

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The full-length fibril gene with modified tryptophan codons was used for expression of fibril protein with or without a terminal (His 6 ) tag in E. coli cells. All the fibril constructs were overexpressed in E. coli BL21 (AI) cells (Invitrogen) by growing the transformants (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. in Luria Bertani (LB) broth supplemented with ampicillin (100 µg/mL final concentration) at 37 °C under shaking conditions until OD 600 reached 0.6. The cultures were then induced with sterile L-arabinose (final concentration 0.2 %) and were grown postinduction at 25 °C for 6 hours. Protein overexpression was confirmed by the presence of high intensity band with molecular weight of ~ 59,000 Da on the SDS-PAGE gel.
Harne S., & Gayathri P. (2021). Characterization of heterologously expressed fibril filaments, a shape and motility determining cytoskeletal protein of the helical bacteriumSpiroplasma.
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