The sequence coding for 6×His tag-labelled ΔMKD1 (ΔMKD1) was amplified by RT-PCR using specific primers (see Supplementary Table S1 ). The amplified PCR products were introduced into SgfI and PmeI sites of the pF3KWG (BYDV) Flexi plasmid (Promega). The ΔMKD1 protein was synthesized using the TNT SP6 High-Yield Wheat Germ Protein Expression System (Promega). In vitro transcription/translation was performed according to the manufacturer’s instructions. The ΔMKD1 protein was purified using a Ni Sepharose High-Performance column (GE Healthcare). MKK1, MKK2, and MKK5 were amplified from cDNA by PCR using specific primers (Supplementary Table S1 ). Amplified fragments of MKK1, MKK2, and MKK5 were cloned into SmaI and NotI (blunt ended) sites of the pGEX6p-1 plasmid (GE Healthcare). MKK1, MKK2, and MKK5 plasmids were transformed into E. coli BL21-CodonPlus (DE3)-RIL (Agilent Technologies). The recombinant proteins glutathione S-transferase (GST)–MKK1, GST–MKK2, and GST–MKK5 were digested by PreScission Protease (GE Healthcare). The resulting MKK1, MKK2, and MKK5 proteins were purified using a glutathione Sepharose 4 Fast Flow column (GE Healthcare).
Ni sepharose high performance column
Manufactured by GE Healthcare
Sourced in United States, Canada
The Ni Sepharose High Performance column is a laboratory equipment designed for protein purification. It utilizes immobilized nickel ions to selectively bind and capture proteins with histidine-tags. The column provides high binding capacity and enhanced performance for efficient protein purification.
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Lab products found in correlation
E coli bl21 codonplus de3 ril, by Agilent Technologies (2 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Pgex 6p 1 plasmid, by GE Healthcare (1 mentions)
Glutathione sepharose 4 fast flow column, by GE Healthcare (1 mentions)
Tnt sp6 high yield wheat germ protein expression system, by Promega (1 mentions)
Prescission protease, by GE Healthcare (1 mentions)
Pdest17, by Thermo Fisher Scientific (1 mentions)
Lr reaction, by Thermo Fisher Scientific (1 mentions)
Pentr d topo vector, by Thermo Fisher Scientific (1 mentions)
Complete ultra, by Roche (1 mentions)
Pdest17 vector, by Thermo Fisher Scientific (1 mentions)
Overnight express autoinduction system 1, by Merck Group (1 mentions)
Bugbuster master mix, by Merck Group (1 mentions)
Quikchange site directed mutagenesis kit, by Agilent Technologies (1 mentions)
Pcdna3, by Genewiz (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Snakeskindialysis tubing 3.5k mwco, by Thermo Fisher Scientific (1 mentions)
Pet29a vector, by Merck Group (1 mentions)
Hitrap heparin hp column, by GE Healthcare (1 mentions)
10 protocols using ni sepharose high performance column
1
Purification and Expression of Arabidopsis MKK Proteins
2
Purification of scFv Antibody Fragments
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Bacterial cells expressing 3F12E7 scFv were suspended in TB buffer (50 mM Tris, pH 7.5) and disrupted by using French Pressure Cell Press (1.6 MPa). The resulting lysate was centrifuged at 10,000 rpm for 10 min. The insoluble fraction was washed twice with TB buffer, incubated with solubilization buffer (50 mM Tris, 300 mM NaCl, 8 M urea, pH 7.5) for 2 h, and then centrifuged at 10,000 rpm. The supernatant was loaded, at a flow rate of 2.5 mL/min, on a Ni-Sepharose high-performance column (GE Healthcare) equilibrated with solubilization buffer and attached to an FPLC system (ÄKTA; GE, USA). Purified scFv was recovered with 200 mM imidazole in elution buffer (50 mM Tris, 300 mM NaCl, 8 M urea, 500 mM imidazole, pH 7.5) and was further analyzed by SDS-PAGE. Protein refolding procedure was performed by 48-h dialysis against water at 4 °C, with water change every 12 h. Samples were filtered through a 0.22-µm filter prior to their in vitro and in vivo application.
3
Recombinant Small Subunit Purification
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The recombinant regulatory (small) subunit was expressed mostly as insoluble inclusion bodies (IBs) under normal growth conditions. It was then purified by using denaturing IMAC followed by on column renaturation as described previously [41] (link). To solubilize the IBs, 5 M urea was incorporated into the composition of lysis buffer [10 mM Tris–HCl, 1 mM EDTA, 50 mM sodium chloride, 5% (v/v) glycerol, 0.5 mg/ml lysozyme, 1 mM SDT and 1 mM DTT, 1 mM TPP, and 10 µM FAD, pH 7.8] and buffer A (20 mM Tris–HCl, 0.5 M NaCl, 5 M urea, 10 mM imidazole, 5% glycerol, 1 mM SDT, and 1 mM DTT, pH 8.0). The cells were lysed using the same procedure as for the catalytic subunit.
After the heat precipitation step (80 °C for 1 h), the CFE was loaded on a Ni Sepharose™ High Performance column (GE Healthcare, QC, Canada) equilibrated with buffer A (20 mM Tris–HCl, 0.5 M NaCl, 5 M urea, 10 mM imidazole, 5% glycerol, 1 mM SDT, and 1 mM DTT, pH 8.0). On column refolding was achieved by applying a linear gradient of urea from 5 M (Buffer A) to 0 M (Buffer B: 20 mM Tris–HCl, 0.5 M NaCl, 10 mM imidazole, 5% glycerol, 1 mM SDT, and 1 mM DTT, pH 8.0), and then the recombinant protein was eluted by applying a gradient of imidazole from 10 mM (Buffer B) to 250 mM (Buffer C: 20 mM Tris–HCl, 0.5 M NaCl, 250 mM imidazole, 5% glycerol, 1 mM SDT, and 1 mM DTT, pH 8.0).
After the heat precipitation step (80 °C for 1 h), the CFE was loaded on a Ni Sepharose™ High Performance column (GE Healthcare, QC, Canada) equilibrated with buffer A (20 mM Tris–HCl, 0.5 M NaCl, 5 M urea, 10 mM imidazole, 5% glycerol, 1 mM SDT, and 1 mM DTT, pH 8.0). On column refolding was achieved by applying a linear gradient of urea from 5 M (Buffer A) to 0 M (Buffer B: 20 mM Tris–HCl, 0.5 M NaCl, 10 mM imidazole, 5% glycerol, 1 mM SDT, and 1 mM DTT, pH 8.0), and then the recombinant protein was eluted by applying a gradient of imidazole from 10 mM (Buffer B) to 250 mM (Buffer C: 20 mM Tris–HCl, 0.5 M NaCl, 250 mM imidazole, 5% glycerol, 1 mM SDT, and 1 mM DTT, pH 8.0).
4
Recombinant Mouse Slx2 Protein Purification
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The entire coding region of mouse Slx2 was subcloned into pENTR/D-TOPO vector (Invitrogen). The full length CDS was recombined to pDEST17 (Invitrogen) using LR reaction (Invitrogen, 11791–020). The 6×His fusion protein was expressed in Escherichia coli strain BL21 and purified using a Ni Sepharose High Performance column (GE Healthcare, 17-5268-01) according to the manufacturer's instructions under denatured condition and on column refolding. Purified protein was used to immunize two healthy rabbits (Shanghai Institutes for Biological Sciences, CAS). Antibody was purified from serum of the rabbits by affinity purification using an antigen-coupled column generated with AminoLink Plus Coupling Resin (Pierce, 20501).
5
Purification of Recombinant Histidine-Tagged PDI
6
Purification of Recombinant Human FGF1 and Mutant
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Wild-type human FGF1 and mutant FGF1 (FGF1-PIGN), shown in Fig. 1A , were purified as described previously [15 (link), 16 (link)]. Briefly, mutations of the human FGF1 gene were introduced using the QuikChange site-directed mutagenesis kit (Agilent Technologies, Santa Clara, CA). The wild-type FGF1 or FGF1-PIGN gene was transferred into the pDEST17 vector (Thermo Fisher Scientific, Waltham, MA), an N-terminal fusion vector that contains a sequence encoding a 6 × His tag, and the pDEST17 expression constructs were transformed into BL21(DE3)pLysS Escherichia coli cells. Protein expression induction was performed using the Overnight Express Autoinduction System 1 (Merck kGaA, Darmstadt, Germany), according to the manufacturer’s instructions. The cell pellets were lysed in BugBuster Master Mix (Merck kGaA) including ethylenediaminetetraacetic acid (EDTA)-free protease inhibitor cocktail (cOmplete ULTRA) (Roche Diagnostics, Mannheim, Germany), and soluble extracts purified using a Ni Sepharose High Performance column (GE Healthcare, Waukesha, WI).
7
Affinity Purification of scFv-MC1
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ScFvMC1 purification was performed as previously published [43 (link)]. Briefly, scFv-MC1 was cloned into the mammalian expression vector pcDNA3.1 (Genewiz, South Plainfield, NJ) and transfected into HEK293T, using Lipofectamine 2000 (Invitrogen, Carlsbad, CA). After 48 h of transfection, the scFv released into the conditioned medium was affinity purified using a Ni-Sepharose High Performance column (GE Healthcare, Port Washington, NY). The efficiency of purification was tested using an immunosorbent assay employed to assess the antigen–binding specificity of the scFvMC1, as previously described [43 (link)]. Starting material, flow through and eluted fractions were tested to check for proper enrichment of the purified material. The purified scFv-MC1 was checked on Coomassie-stained SDS-PAGE gel for proper molecular weight.
8
Recombinant Destabilase Production in Human Cells
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The preparation of recombinant destabilase was described in detail earlier37 (link). Briefly, DNA fragments encoding destabilase isoform Ds2 (UniProt ID Q25091) were synthesized from oligonucleotides. The plasmid pcDNA3.4-Dest2 for the expression of the destabilase gene in the human cell line Expi293F was constructed using a pcDNA3.4-TOPO TA Cloning Kit (Invitrogen, USA). The transient human cell line Expi293F producing destabilase was generated using an Expi293F Expression System Kit (Life Technologies, USA). The cells were transfected by ExpiFectamine 293 transfection agent with pcDNA3.4-Dest2 plasmid followed by incubation for 120 h, the enzyme was secreted into the culture medium. The presence of C-terminal 6xHisTag allowed for the concentration and purification of the target protein using metal chelate chromatography on Ni Sepharose High Performance column (GE Healthcare). The yield of pure destabilase was 20 mg per 1 l of the human cell line culture. Additional purification step was performed using CM Sepharose Fast Flow media (GE Healthcare, USA). The solution of purified destabilase was dialyzed (SnakeSkin Dialysis Tubing 3.5 K MWCO, Thermo Fisher Scientific) against 5 mM Na2HPO4 at pH 5.0.
9
Purification of AtNFXL1ΔNΔZn Protein
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The AtNFXL1ΔNΔZn fragment (2341–3567 bp) was amplified by PCR from cDNA using specific primers (see Supplementary Table S1 at JXB online). The amplified fragment of AtNFXL1ΔNΔZn was cloned into the NdeI and SalI sites of the pET-29a vector (Merck KGaA). The plasmids were transformed into E. coli BL21-CodonPlus (DE3)-RIL (Agilent Technologies). The 6×Histidine (His) tag-labelled AtNFXL1ΔNΔZn protein (His–AtNFXL1ΔNΔZn protein) was purified using a Ni Sepharose High Performance column (GE Healthcare). SDS-PAGE and immunoblotting were carried out as previously described (Asano et al., 2004 (link)). The anti-AtNFXL1C antibody was generated in rabbit and purified using antigen (His–AtNFXL1ΔNΔZn protein)-coupled HiTrapTM NHS (N-hydroxysuccinimide)-activated HP (high performance; GE Healthcare). Then, an AtNFXL1-containing protein complex was purified using anti-AtNFXL1C antibody coupled to HiTrapTM NHS-activated HP.
10
LwaCas13a Protein Purification Protocol
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Escherichia coli Rosetta 2(DE3) was transformed with the pET-LwaCas13a plasmid, and the cells were cultured at 37 °C in 100 mL TB medium, supplemented with chloramphenicol and kanamycin. When the OD600 reached 0.6–0.8, the bacterial culture was cooled on ice for 10 min, and then further cultured at 20 °C for 20 h with 0.1 mM IPTG. The E. coli cells were collected, suspended in 5 mL buffer A (20 mM Tris-HCl (pH 8.0), 1 M NaCl, and 20 mM imidazole), and disrupted by a sonicator (Q500, QSONICA). After centrifugation at 15,000 rpm for 15 min, the supernatant was loaded onto a Ni-Sepharose High Performance column (GE Healthcare), equilibrated with buffer A. The protein was eluted with buffer B (20 mM Tris-HCl (pH 8.0), 300 mM NaCl, and 400 mM imidazole). The protein was loaded onto a HiTrap Heparin HP column (1 mL; GE Healthcare), equilibrated with buffer C (20 mM Tris-HCl (pH 8.0) and 300 mM NaCl). The protein was eluted with a linear gradient of 0.3–2 M NaCl. The fractions were analyzed by SDS-PAGE, and peak fractions were pooled. The protein concentration was determined according to the A280 value measured by a NanoDrop spectrophotometer (Thermo Scientific). Aliquots of the purified LwaCas13a protein were quickly frozen in liquid nitrogen and stored at −80 °C until measurements.
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