A plasmid bearing 6XHis-CnRho1 (CNAG_03315) was transformed into One Shot BL21 (DE3) E. coli cells (Invitrogen). A single colony was inoculated into LB + Ampicillin and grown overnight at 37 °C and then subcultured in 250 mL LB + Ampicillin for 3 h at 30 °C. Then, 50 mL of the culture was collected for the uninduced fraction, and then, the culture was induced with 0.5 mM isopropylthio-β-galactoside (IPTG; Invitrogen) and continued incubation for 1 h. Cell pellets were lysed in Qiagen native lysis buffer with benzonase, lysates were applied to the Ni-NTA column (Ni-NTA Fast Start Kit, Qiagen), and purification was performed following the manufacturers’ instructions. Protein was eluted with two rounds of 750 μL of elution buffer. Each fraction was run on an SDS-PAGE gel. Rho GTPase activity was measured using the GTPase-Glo (Promega) Assay kit. Then, 800 ng of Rho1 per reaction was preincubated with 25 μg/mL 1 or DMSO for 30 min at room temperature (RT), and then, the GTPase reaction was started with 1 μM GTP and 1 mM DTT per the manufacturers’ instructions. The reactions were stopped at indicated time points and developed per manufacturers’ instructions. The luminescence was read with a 100 ms integration time.
One shot bl21 de3 e coli cells
Manufactured by Thermo Fisher Scientific
One Shot BL21 (DE3) E. coli cells are competent bacterial cells designed for high-efficiency transformation and protein expression. They are derived from the BL21 (DE3) strain, which is widely used for recombinant protein production in Escherichia coli.
Automatically generated - may contain errors
Lab products found in correlation
Histrap hp column, by GE Healthcare (2 mentions)
Ni nta bead, by Qiagen (2 mentions)
Coomassie brilliant blue, by Bio-Rad (2 mentions)
Ni nta fast start kit, by Qiagen (1 mentions)
Isopropylthio β galactoside iptg, by Thermo Fisher Scientific (1 mentions)
Isopropyl β d 1 thiogalactopyranoside (iptg), by Teknova (1 mentions)
4 protocols using one shot bl21 de3 e coli cells
1
Purification and Activity Assay of Rho1 GTPase
2
Purification and Refolding of Hrgβ1 Protein
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Hrgβ1
expression
plasmid was transformed into chemically competent One Shot BL21 (DE3) E. coli cells (Invitrogen, C600003). After selection
and growth of a single clone in LB, expression of Hrgβ1 was
induced by 40 μM IPTG (Teknova, I3305). After growth at 225
RPM for 3 h at 37 °C, the centrifuged pellet was frozen at −80
°C, thawed at 4 °C, resuspended in lysis buffer (50 mM Tris
HCl, pH 8.0, 500 mM NaCl, 5% (v/v) glycerol, 5 mM imidazole, 0.025%
(v/v) Tween 20, 0.01% (w/v) sodium azide) with an EDTA-free protease
inhibitor tablet, sonicated, and clarified at 19,000 × g for 60 min at 4 °C. The pellet was further extracted
with lysis buffer including 6 M guanidine-HCl. The solution was frozen
at −80 °C and then thawed rotating end-over-end at 4 °C.
Ni2+-NTA purification was used to bind the Hrgβ1
denatured lysate. To refold on the resin, bound protein was sequentially
washed with lower amounts of guanidine-HCl (6 to 0 M) and higher amounts
of imidazole (10 to 50 mM). After elution with 250 mM imidazole, recovered
Hrgβ1 was buffer-exchanged into 25 mM HEPES pH 8.0, 300 mM NaCl,
10% (v/v) glycerol, and 0.01% (w/v) sodium azide.
expression
plasmid was transformed into chemically competent One Shot BL21 (DE3) E. coli cells (Invitrogen, C600003). After selection
and growth of a single clone in LB, expression of Hrgβ1 was
induced by 40 μM IPTG (Teknova, I3305). After growth at 225
RPM for 3 h at 37 °C, the centrifuged pellet was frozen at −80
°C, thawed at 4 °C, resuspended in lysis buffer (50 mM Tris
HCl, pH 8.0, 500 mM NaCl, 5% (v/v) glycerol, 5 mM imidazole, 0.025%
(v/v) Tween 20, 0.01% (w/v) sodium azide) with an EDTA-free protease
inhibitor tablet, sonicated, and clarified at 19,000 × g for 60 min at 4 °C. The pellet was further extracted
with lysis buffer including 6 M guanidine-HCl. The solution was frozen
at −80 °C and then thawed rotating end-over-end at 4 °C.
Ni2+-NTA purification was used to bind the Hrgβ1
denatured lysate. To refold on the resin, bound protein was sequentially
washed with lower amounts of guanidine-HCl (6 to 0 M) and higher amounts
of imidazole (10 to 50 mM). After elution with 250 mM imidazole, recovered
Hrgβ1 was buffer-exchanged into 25 mM HEPES pH 8.0, 300 mM NaCl,
10% (v/v) glycerol, and 0.01% (w/v) sodium azide.
3
Purification of Recombinant Plant Proteins
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N-terminally 6xHis-tagged AtPNP-A and CAT2 proteins were expressed in BL21 (DE3) One Shot E. coli cells (Life Technologies), purified by affinity chromatography with Ni–NTA beads (Qiagen) and HisTrap HP column (GE Healthcare Lifesciences) as described in21 (link). The purity of preparations was verified on 12.5% SDS-PAGE, stained with Coomassie Brilliant Blue (Bio-Rad). Protein identities were confirmed in MS analysis, and protein concentration was determined according to the method of Bradford using BSA as a standard.
4
Recombinant AtPNP-A and RCA Protein Expression
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Expression of the N-terminally 6xHis-tagged AtPNP-A and RCA proteins was conducted in BL21 (DE3) One Shot E. coli cells (Life Technologies). The recombinants were purified by affinity chromatography with Ni-NTA beads (Qiagen, Venlo, the Netherlands) and HisTrap HP column (GE Healthcare, Chicago, IL, USA) as previously described [25 (link)]. The purity of protein preparations was verified on 12.5% SDS-PAGE stained with Coomassie Brilliant Blue (Bio-Rad, Hercules, CA, USA). Identity of the recombinants was confirmed in MS analysis, and protein concentration was determined by the Bradford method using bovine serum albumin (BSA) as a standard.
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