Bl21 codonplus de3 ripl

Manufactured by Agilent Technologies

Sourced in United States

The BL21-CodonPlus (DE3)-RIPL is a strain of E. coli cells designed for high-level recombinant protein expression. It contains additional copies of the argU, ileY, and leuW tRNA genes to enhance the expression of heterologous proteins.

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Lab products found in correlation

Ni nta, by Qiagen (6 mentions) Cnbr activated sepharose, by GE Healthcare (5 mentions) Quikchange lightning site directed mutagenesis kit, by Agilent Technologies (5 mentions) Protein purification kit, by Takara Bio (4 mentions) Gateway system, by Thermo Fisher Scientific (4 mentions) Gateway pet dest42 vector, by Thermo Fisher Scientific (3 mentions) Hispur cobalt resin kit, by Thermo Fisher Scientific (3 mentions) Pcdna3.1 vector, by Thermo Fisher Scientific (3 mentions) Pgem 3z vector, by Promega (3 mentions) Lipofectamine 3000, by Thermo Fisher Scientific (3 mentions) 4d nucleofector system, by Lonza (3 mentions) Protease inhibitor cocktail, by Merck Group (3 mentions) Histrap hp column, by GE Healthcare (2 mentions) Ni nta agarose, by Qiagen (2 mentions) Recombinant flag gpnmb, by OriGene (2 mentions) Gst egfr, by Active Motif (2 mentions) Recombinant active caspase 1, by R&D Systems (2 mentions) Transcend non radioactive translation detection system, by Promega (2 mentions) Tnt quick coupled transcription translation kit, by Promega (2 mentions) Hif 1α, by Novus Biologicals (2 mentions)

Close spelling variants of this product

BL21-CodonPlus (DE3)-RIPL cells (21 citations) BL21(DE3) (20 citations) BL21-CodonPlus (DE3)-RIPL competent cells (18 citations) BL21(DE3)-RIPL (5 citations) BL21-CodonPlus (DE3)-RIPL chemically competent cells (3 citations) E. coli BL21-CodonPlus(DE3)-RIPL (3 citations) E. coli BL21-CodonPlus (DE3)-RIPL cells (2 citations)

52 protocols using bl21 codonplus de3 ripl

Affinity Purification of Recombinant Proteins

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His-MBP-TMUB1 was purified using Ni-NTA Sefinose Resin (Sangon Biotech) after being expressed in Escherichia coli strain BL21-CodonPlus (DE3)-RIPL (Agilent Technologies). GST-PD-L1 was isolated using GST magnetic beads (Sangon Biotech) after being produced in Escherichia coli strain BL21-CodonPlus (DE3)-RIPL (Agilent Technologies) (Sangon Biotech). HUWE1-Flag was isolated using Flag-M2 magnetic beads (Sigma) from the cell lysate of HEK-293T cells with HUWE1-Flag overexpression. The concentration and purity of recombinant proteins were determined using SDS-PAGE and Coomassie staining using BSA as a reference.
GST-tagged protein with the His-tagged protein or Flag-tagged protein (1–3 µg) wer incubated with GST magnetic beads (Sangon Biotech) in 500 µl of binding buffer (50 mM Tris-HCl at pH 7.9, 10% glycerol, 100 mM KCl, 5 mM MgCl2, 10 mM β-mercaptoethanol and 0.1% NP-40) for 2 h at 4 °C with gentle rotation. The beads were then washed with NETN buffer 3 times for 5 min each time at 4 °C with rotation. Subsequently, the beads were eluted in 50 μl 2× SDS loading buffer, and the eluted protein complexes were detected by IB.

Shi C., Wang Y., Wu M., Chen Y., Liu F., Shen Z., Wang Y., Xie S., Shen Y., Sang L., Zhang Z., Gao Z., Yang L., Qu L., Yang Z., He X., Guo Y., Pan C., Che J., Ju H., Liu J., Cai Z., Yan Q., Yu L., Wang L., Dong X., Xu P., Shao J., Liu Y., Li X., Wang W., Zhou R., Zhou T, & Lin A. (2022). Promoting anti-tumor immunity by targeting TMUB1 to modulate PD-L1 polyubiquitination and glycosylation. Nature Communications, 13, 6951.

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Recombinant Archease Protein Purification

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Human Archease isoform 1 (UniProt A8K0B5; note the deviating translation start sites from UniProt Q8IWT0 by twelve additional residues) was expressed and purified as described previously^{10 (link)}. In short, the fusion protein with an N-terminal His₆ tag followed by a thrombin protease cleavage site was expressed in E. coli BL21-CodonPlus (DE3)-RIPL (Agilent Technologies) cells. The clarified lysate was applied to a 5 mL Ni-NTA column (Cytiva). After tag removal (optional) with thrombin (Cytiva), the sample was re-applied onto the Ni-NTA column to remove the affinity tag. The flow-through fraction was concentrated using a centrifugal filter (Amicon Ultra, MWCO 10 kDa, Sigma) and purified by size-exclusion chromatography with buffer containing 25 mM HEPES, pH 7.5, 100 mM NaCl₂, 5% Glycerol, 1 mM TCEP on HiLoad16-600 Superdex 75 pg column (Cytiva). Archease containing peak fractions were concentrated, flash-frozen in liquid nitrogen and stored at −80 °C. We determined a 260/280 nm ratio of 0.56 for the final protein preparation indicating the absence of nucleic acid contaminants (see Source Data).

Gerber J.L., Morales Guzmán S.I., Worf L., Hubbe P., Kopp J, & Peschek J. (2024). Structural and mechanistic insights into activation of the human RNA ligase RTCB by Archease. Nature Communications, 15, 2378.

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Recombinant Expression and Purification of Human ProRS

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Wild-type or P1482T mutant ProRS fragment of human EPRS1 (aa 930-1512) was subcloned in pTRC-HisB (Invitrogen) for N-terminal 6X-His tagging, and sequence verified. Recombinant protein was expressed in BL21 Codon-plus (DE3)RIPL (Agilent) strain as described^{21 (link),103 (link)}. Briefly, protein was induced with isopropyl β-D-1-thiogalactopyranoside (200 μM) at 37 °C, and cells harvested by centrifugation 4–6 h post-induction. The pellet was resuspended in purification buffer containing 50 mM Tris-HCl, pH 8.0, 100 mM NaCl,10% glycerol,1 mg/ml lysozyme, protease inhibitors, and 10 mM imidazole, and sonicated on ice for 20 min. Lysate was cleared by centrifugation at 26,000 × g for 45 min, and purified using HisTrap HP column (GE Life Sciences, Pittsburgh, PA). Protein oligomeric state was determined using a Superdex 200 size-exclusion column (GE Life Sciences) pre-calibrated with Bio-Rad gel-filtration standards (Bio-Rad) in purification buffer on an NGC Chromatography System with ChromLab v5.0.2.11 (Bio-Rad). ProRS aminoacylation activity was confirmed as described^{21 (link)}.

Khan D., Ramachandiran I., Vasu K., China A., Khan K., Cumbo F., Halawani D., Terenzi F., Zin I., Long B., Costain G., Blaser S., Carnevale A., Gogonea V., Dutta R., Blankenberg D., Yoon G, & Fox P.L. (2024). Homozygous EPRS1 missense variant causing hypomyelinating leukodystrophy-15 alters variant-distal mRNA m6A site accessibility. Nature Communications, 15, 4284.

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Expression and Purification of CtISWI ATPase Fragment

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Escherichia coli BL21-CodonPlus(DE3)-RIPL (Agilent Technologies, Wilmington, DE, USA) was used for expression of the recombinant CtISWI_77-Δ-722 ATPase fragment. Escherichia coli cells harboring the CtISWI_77-Δ-722 ATPase fragment expression plasmid were grown at 37°C in 1× LB Broth (IPM Scientific) with 100 μg ml⁻¹ Ampicillin until OD₆₀₀ reached around 1.0. At this point, recombinant protein expression was induced by adding 0.3 mM IPTG (isopropyl β-d-1-thiogalactopyranoside, final concentration) for 18–20 h at 14°C. The cells were harvested at 4,000 RPM for 10 min, then washed twice with 40 ml wash buffer containing 20 mM Tris–HCl pH 8.0, 500 mM NaCl and 2 mM β-ME. Cells were resuspended in 40 ml binding buffer containing 20 mM Tris–HCl pH 8.0, 500 mM NaCl, 20 mM imidazole, 5 mM β-ME, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 U ml⁻¹ Benzonase Nuclease (Sigma-Aldrich, Saint Louis, MO, USA) and 10 μg ml⁻¹ Ribonuclease A (Sigma-Aldrich, Saint Louis, MO), followed by sonication on ice for a total of 10 min with a pulse of 5 s on, and 10 s off, and centrifugation at a speed of 35,000 RPM for 2 h and at 4°C. The protein was first purified using Ni-NTA agarose (QIAGEN, Valencia, CA, USA) following the protocol provided by the manufacturer and further purified by one round of size-exclusion chromatography (SEC) in a buffer containing 20 mM Tris–HCl pH 7.4, 200 mM NaCl, and 5 mM β-ME.

Chittori S., Hong J., Bai Y, & Subramaniam S. (2019). Structure of the primed state of the ATPase domain of chromatin remodeling factor ISWI bound to the nucleosome. Nucleic Acids Research, 47(17), 9400-9409.

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Overexpression and Purification of MacUDG and MacUDG-like

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MacUDG and MacUDG-like were overproduced in E. coli BL21 CodonPlus (DE3)-RIPL (Agilent Technologies) cells carrying the respective plasmids pET-MA_RS11760 and pET-MA_RS18745. The cells were grown with shaking in 1 L of LB medium, containing 50 µg/mL ampicillin and 34 µg/mL chloramphenicol at 37 °C until the OD₆₀₀ reached 0.6 for MacUDG-like and 0.4 for MacUDG. IPTG was added to a final concentration of 0.1 mM, and the cells were further grown at 18 °C overnight for MacUDG-like and at 16 °C overnight for MacUDG. The proteins were prepared in nearly the same manner as described for MacExoIII. The eluted protein fractions were stored at −20 °C with 50% glycerol or at −80 °C. The purities of the proteins were evaluated by 15% SDS-PAGE followed by CBB staining. The protein concentrations were calculated by measuring the absorbance at 280 nm. The theoretical molar extinction coefficients of N-terminal 6× His-tagged MacUDG-like and MacUDG are 21,110 and 21,930, respectively.

Shiraishi M., Ishino S., Heffernan M., Cann I, & Ishino Y. (2018). The mesophilic archaeon Methanosarcina acetivorans counteracts uracil in DNA with multiple enzymes: EndoQ, ExoIII, and UDG. Scientific Reports, 8, 15791.

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Polyclonal Antibodies Generation for FBXO47

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Polyclonal antibodies against mouse FBXO47 C-terminal (aa272-451) were generated by immunizing rabbits and a guinea pig. FBXO47 middle region (aa174-316) were generated by immunizing a rabbit. His-tagged recombinant proteins of FBXO47 middle region (aa174-316) and C-terminal (aa272-451) were produced by inserting cDNA fragments in-frame with pET19b and pET28c (Novagen) respectively in E. coli strain BL21-CodonPlus (DE3)-RIPL (Agilent), solubilized in a denaturing buffer (6 M HCl-Guanidine, 20 mM Tris-HCl pH 7.5) and purified by Ni-NTA (QIAGEN) under denaturing conditions. The antibodies were affinity-purified from the immunized serum with immobilized antigen peptides on CNBr-activated Sepharose (GE healthcare).

Tanno N., Takemoto K., Takada-Horisawa Y., Shimada R., Fujimura S., Tani N., Takeda N., Araki K, & Ishiguro K.I. (2022). FBXO47 is essential for preventing the synaptonemal complex from premature disassembly in mouse male meiosis. iScience, 25(4), 104008.

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Fission Yeast Protein Expression in E. coli

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Fission yeast Psc3, Wapl and RPA were expressed in the E. coli strain BL21-CodonPlus(DE3)-RIPL (Agilent Technologies). The genotype is: E. coli B F^-ompT hsdS(r_B^-m_B^-) dcm⁺Tet^rgal λ(DE3) endA Hte [argU proL BBCamr] [argU ileY leuW Strep/Specr].

Murayama Y., Samora C.P., Kurokawa Y., Iwasaki H, & Uhlmann F. (2018). Establishment of DNA-DNA Interactions by the Cohesin Ring. Cell, 172(3), 465-477.e15.

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Recombinant Protein Expression and Purification

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Recombinant proteins were expressed in E.coli strain BL21-CodonPlus^® (DE3)-RIPL (Agilent Technologies) and purified using Protein Purification Kit (Clontech). Recombinant Flag-GPNMB and PHD1 were purchased from Origene. GST-EGFR was purchased from Active Motif, HIF1α and BRK were purchased from Novus Biologicals. LRRK2 was purchased from SignalChem. Recombinant active Caspase-1 was purchased from R&D Systems. In vitro translation of LINK-A was conducted using TnT^® Quick Coupled Transcription/Translation Kit and detection was performed using Transcend™ Non-Radioactive Translation Detection System (Promega).

Lin A., Li C., Xing Z., Hu Q., Liang K., Han L., Wang C., Hawke D.H., Wang S., Zhang Y., Wei Y., Ma G., Park P.K., Zhou J., Zhou Y., Hu Z., Zhou Y., Marks J.R., Liang H., Hung M.C., Lin C, & Yang L. (2016). The LINK-A lncRNA Activates Normoxic HIF1α Signaling in Triple-negative Breast Cancer. Nature cell biology, 18(2), 213-224.

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Cloning and Expression of MotA Protein from Aquifex aeolicus

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The full-length motA gene from A. aeolicus (motA^Aa) was cloned into a pColdI vector (Takara Bio Inc., Kusatsu, Japan) as previously described24 (link). MotA^Aa protein was expressed with an N-terminal histidine tag in E. coli BL21-CodonPlus(DE3)-RIPL (Agilent Technologies, Santa Clara, CA, USA) cells. The cells were grown in 1.5 L of SB medium [1.2% (w/v) Bacto tryptone, 2.4% (w/v) Bacto yeast extract, 0.5% (w/v) glycerol, 1.25% (w/v) K₂HPO₄, 0.38% (w/v) KH₂PO₄] containing 100 μg/mL ampicillin, at 37 °C, to an OD₆₆₀ of 0.6–0.8, and 0.5 mM isopropyl-β-d-thiogalactopyranoside (IPTG) was subsequently added to the culture after cooling on ice for 30 min before the culture was prolonged for about 20 h at 16 °C. Cells were collected by centrifugation (7,000 × g) and then resuspended in 50 mL of TN buffer [50 mM Tris-HCl (pH 8.0], 200 mM NaCl) containing half a protease inhibitor cocktail tablet (Roche diagnostics) and about 10 mg of lysozyme (Wako, Osaka, Japan). Cells were then disrupted by sonication and ultracentrifuged at 100,000 × g for 30 min, and the pellet (membrane fraction) resuspended in TN buffer.

Takekawa N., Terahara N., Kato T., Gohara M., Mayanagi K., Hijikata A., Onoue Y., Kojima S., Shirai T., Namba K, & Homma M. (2016). The tetrameric MotA complex as the core of the flagellar motor stator from hyperthermophilic bacterium. Scientific Reports, 6, 31526.

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Sf9 Cell Culture and Recombinant Protein Expression

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Sf9 (Spodoptera frugiperda) cells were maintained as a
suspension culture in Sf-900II serum-free medium (SFM) (Thermo Fisher
Scientific) at 27°C according to the manufacturers recommendations. Cells
were shaken at ~150 rpm on a rotary shaker. The culture density was
maintained between 1.0 × 10⁶ ~ 8.0 ×
10⁶ cells/mL during routine passage. Infection of bacmid was
performed at 1.0 × 10⁶ cells/mL. Protein expression was
performed by infection of a culture at ~2.0 × 10⁶cells/mL with baculovirus at a 1:100 volume:volume ratio. Cells were not
authenticated. The sex of the cells is unknown.
BL21-CodonPlus (DE3)-RIPL (Agilent) E. Coli transformed
with each plasmid were grown at 37°C in LB medium with shaking at
~ 200 rpm. Cells were not authenticated.

Chiba K., Ori-McKenney K.M., Niwa S, & McKenney R.J. (2022). Synergistic autoinhibition and activation mechanisms control kinesin-1 motor activity. Cell reports, 39(9), 110900.

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