CRP1 and CRP4 cDNAs were amplified by PCR and inserted into the prokaryotic pET19b-10×His-SUMO expression vector. CRP cDNAs were inserted in-frame into the expression vector, allowing for creation of N-terminal 10×His-SUMO tags on the CRPs. The pET19b-10×His-SUMO-CRP1 and pET19b-10×His-SUMO-CRP4 expression vectors were transformed into BL21(DE3)pLysS competent cells (Promega, Madison, WI, USA). Transformants were grown overnight and then used for protein inductions. After 4 h of induction at 37 °C, cells were pelleted and lysed in 1× FastBreak cell lysis reagent according to the manufacturer’s instructions (Promega, Madison, WI, USA). Extracts were sonicated 2 min to ensure complete bacterial cell lysis (10 sec pulses). Following sonication, crude extracts were clarified by centrifugation at 16,000× g for 10 min at 4 °C. Supernatants were filtered through a 0.2 micron filter and then used for affinity chromatography purification.
Fastbreak cell lysis reagent
Manufactured by Promega
Sourced in United States
FastBreak Cell Lysis Reagent is a solution designed to efficiently lyse a variety of cell types, facilitating the release of cellular contents for subsequent analysis or purification. The reagent is formulated to disrupt cell membranes while preserving the integrity of the released macromolecules.
Automatically generated - may contain errors
Lab products found in correlation
Ni nta agarose, by Qiagen (2 mentions)
Bl21 de3 plyss competent cells, by Promega (1 mentions)
Edta free protease inhibitor tablet, by Roche (1 mentions)
Econo pac chromatography column, by Bio-Rad (1 mentions)
Amicon ultra 15, by Merck Group (1 mentions)
Bio safe coomassie stain, by Bio-Rad (1 mentions)
Complete mini edta free protease inhibitor cocktail, by Roche (1 mentions)
Dual luciferase reporter assay system, by Promega (1 mentions)
Withlipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Isopropyl β d 1 thiogalactopyranoside (iptg), by Merck Group (1 mentions)
Terrific broth, by Thermo Fisher Scientific (1 mentions)
Glutathione sepharose 4b, by GE Healthcare (1 mentions)
Hitrap desalting column, by GE Healthcare (1 mentions)
Gstrap 4b column, by GE Healthcare (1 mentions)
Mouse ifn α, by R&D Systems (1 mentions)
Biacore x instrument, by GE Healthcare (1 mentions)
Hitrap, by Cytiva (1 mentions)
8 protocols using fastbreak cell lysis reagent
1
Recombinant Production and Purification of CRP Proteins
2
Affinity Purification of Histidine-Tagged Proteins
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Cell pellets were re-suspended in 90 μl of buffer prepared by adding one cOmplete EDTA-free Protease Inhibitor tablet (Roche) to 50 ml of 50 mM Tris pH 8, 0.5 M NaCl, 5 mM imidazole. An aliquot (60 μl) of 1× FastBreak Cell Lysis Reagent (Promega) was added and the lysate was incubated at 37°C for 15 min and at 70°C for 15 min, before centrifugation through a Millipore 96-well filter plate. Clarified lysates were combined with 60 μl Ni-NTA agarose (Qiagen) and incubated with shaking at room temperature for 2 h. After collecting the agarose resins using a fresh filter plate, resins were washed two times with 200 μl wash buffer (50 mM Tris pH8, 0.5 M NaCl, 20 mM imidazole) and eluted with 80 μl of 50 mM Tris pH8, 0.5 M NaCl, 200 mM imidazole.
3
Purification of Cas9 and Acr Proteins
4
Automated High-Throughput Protein Expression
5
Measuring Wnt Signaling Activity in Hone1 Cells
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TOP flash and TCF4 were designed and synthesized by Sangon Biotech Co., Ltd.
Hone1 cells were cultured in 96 well plates (20 000 cells/50 μL) and transfected
with 20 ng of TOP flash. Following that, cells were transfected with
Lipofectamine™ 2000 (Invitrogen, Carlsbad, CA) as the manufacturer’s protocol.
The cells were lysed using FastBreak™ Cell Lysis Reagent (Promega Corporation,
Madison, WI, USA) and the luciferase activities were assayed by Dual-Luciferase
Reporter assay system (Promega Corporation).
Hone1 cells were cultured in 96 well plates (20 000 cells/50 μL) and transfected
with 20 ng of TOP flash. Following that, cells were transfected with
Lipofectamine™ 2000 (Invitrogen, Carlsbad, CA) as the manufacturer’s protocol.
The cells were lysed using FastBreak™ Cell Lysis Reagent (Promega Corporation,
Madison, WI, USA) and the luciferase activities were assayed by Dual-Luciferase
Reporter assay system (Promega Corporation).
6
Recombinant DDX1 and DDX3 Protein Purification
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Recombinant DDX1 and DDX3 proteins were purchased from OriGene. The recombinant Nullbasic-FLAG-V5-6xHis was produced in E. coli strain BL21-AI (Invitrogen) transformed by pDEST42-Nullbasic-FLAG. A 200 ml culture was grown in Luria broth to log phase and induced with 0.2% arabinose and 1 mM IPTG. After 4 hours the E. coli was collected by centrifugation and immediately processed. 1× FastBreak™ Cell Lysis Reagent (Promega) was added to the cell pellet for 15 min and the lysate was mixed with 2 ml of Chelating Sepharose beads (GE Healthsciences) for 1 min. The beads were placed into a column and wash buffer (100 mM HEPES pH7.5, 10 mM imidazole) was applied 6 times. Two ml of elution buffer (100 mM HEPES pH7.5, 500 mM imidazole) was added and after 1 min was collected. The eluted protein was dialysed twice for 90 minutes each in 1 liter of storage buffer (20% glycerol v/v, 50 mM Tris–HCl pH8.0, 1 mM ZnCl2, adding fresh 2 mM 1,4-Dithioerythritol every 30 minutes). The protein was stored in aliquots under liquid nitrogen.
7
Purification of GST-GFP-P4M Fusion Protein
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BL21-competent cells were transformed with GST-GFP-P4M. A single transformed colony was inoculated in 100 ml of Terrific Broth (Invitrogen) supplemented with 0.1% glycerol and incubated overnight at 37°C. The next day, 20 ml of the overnight culture was inoculated into 400 ml of Terrific Broth with glycerol and incubated for 2 h at 37°C. After the incubation, isopropyl β-d-1-thiogalactopyranoside (Sigma-Aldrich) was added (final concentration 0.25 mM), and the culture was incubated at 30°C for 4 h. After the incubation, cells were lysed using FastBreak cell lysis reagent (Promega) according to the manufacturer’s instructions. The cell lysate was then centrifuged and the supernatant collected. The presence of GST-GFP-P4M was confirmed by Western blot using an α-GFP antibody (GFP(B-2); sc-9996; Santa Cruz Biotechnology, Dallas, TX). The supernatant was then incubated with Glutathione Sepharose 4B (GE Healthcare; previously washed three times with binding buffer, phosphate-buffered saline [PBS], pH 7.3) overnight at 4°C. The solution was then centrifuged and washed three times with binding buffer. Finally, the pellet was incubated in elution buffer (50 mM Tris-HCl plus 10 mM reduced glutathione, pH 8.0) for 30 min at room temperature, and the elution was collected. The presence of the purified GST-GFP-P4M was confirmed by immunoblotting with anti-GFP antibody (GFP(B-2).
8
Purification and Analysis of Mouse IFN-α
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Recombinant proteins of mouse sortilin and cytokines except for mouse IFN-α were purchased from R&D. GST-fused mouse IFN-α was purified as follows: the plasmid pGEX-IFNA2 was introduced into Escherichia coli strain Rosetta 2(DE3) (Novagen, Madison, WI). Plasmid-bearing cells were initially grown aerobically at 37 °C on L medium46 containing 100 μg/ml ampicillin. Expression of recombinant proteins was then induced by the addition of 0.2 mM IPTG at 20 °C for 18 h. Cell lysates were prepared with FastBreak Cell Lysis Reagent (Promega, Madison, WI). Isolated supernatants were purified with a GSTrap 4B column (GE Lifesciences). Eluted protein fractions were then desalted through a HiTrap Desalting column (GE Lifesciences) and fractions containing purified proteins were concentrated with Vivaspin 20 spin columns (GE Lifesciences). Concentrated proteins were stored at −80 °C until use. Surface plasmon resonance analysis was performed as described47 (link) on a Biacore X instrument (GE Lifesciences). Kinetic parameters were determined using BIAevaluation software.
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