E. coli cells harvested from 1 L LB medium were resuspended in 100 mL Buffer A (50 mM Tris-HCl, 20 mM imidazole, and 500 mM NaCl; pH 7.4), followed by three passes through the JN-3000PLUS cell crusher (Jnbio, Guangzhou, China), and centrifuged to remove cell debris. The supernatants were collected after centrifugation (15 000 rpm, 30 min, 4°C) and applied to a column loaded with 3 mL Ni-NTA BEADS (Smart-Life Sciences, Changzhou, China). The Ni-NTA BEADS were washed three times with 15 mL Buffer A and eluted with 15 mL Buffer B (50 mM Tris-HCl, 500 mM imidazole, and 500 mM NaCl; pH 7.4). The elution was centrifuged using Amicon Ultra-15 Centrifugal Filters (Millipore, Burlington, MA) to concentrate enzymes and exchange buffer into Buffer C (50 mM Tris-HCl, 50 mM NaCl, and 10% glycerol; pH 7.4). The protein concentration was determined with a BCA assay kit (Biosharp, Hefei city, China). The purified enzymes were aliquoted and stored at –80°C. All steps were performed on ice with pre-cooled buffers.
Ni nta beads
Manufactured by Smart-Lifesciences
Sourced in China
Ni-NTA beads are a type of affinity chromatography resin used for the purification of histidine-tagged recombinant proteins. The beads contain nickel ions (Ni2+) that bind to the histidine residues on the target protein, allowing it to be separated from other cellular components during the purification process.
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Lab products found in correlation
Amicon ultra 15 centrifugal filter, by Merck Group (1 mentions)
Bca assay kit, by Biosharp (1 mentions)
Anti his antibody, by Santa Cruz Biotechnology (1 mentions)
Mono q 5 50 gl column, by GE Healthcare (1 mentions)
Dextrin beads 6ff, by Smart-Lifesciences (1 mentions)
Hiload 16 60 superdex 200 size exclusion column, by GE Healthcare (1 mentions)
Emsa chemiluminescence kit, by Beyotime (1 mentions)
Glutathione beads, by Smart-Lifesciences (1 mentions)
Superdex 200 10 300 gl size exclusion column, by Cytiva (1 mentions)
Dextrin beads, by Smart-Lifesciences (1 mentions)
Bac to bac baculovirus expression system, by Thermo Fisher Scientific (1 mentions)
3 kda ultrafiltration tube, by Merck Group (1 mentions)
Superdex 200 increase 10 300 column, by GE Healthcare (1 mentions)
Bradford method, by Sangon (1 mentions)
Streptavidin sepharose bead, by GE Healthcare (1 mentions)
10 kda ultrafiltration tube, by Merck Group (1 mentions)
Pet28a vector, by Genewiz (1 mentions)
Superdex 75 increase 10 300 gl, by GE Healthcare (1 mentions)
10 protocols using ni nta beads
1
Purification of Recombinant Proteins from E. coli
2
Expression and Purification of SFTS Virus Glycoproteins
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Amino acid sequence of Gn was analyzed by TMHMM Server v.2.0. Both the hydrophobic N- and C-termini of Gn or Gc were removed for a soluble expression. The truncated sequence of Gn expands from amino acid residue D20 to C447, whereas the truncated sequence of Gc expands from amino acid residue C563 to N1026, based on Genbank accession number: YP_006504094.1 for the membrane glycoprotein polyprotein [SFTS virus HB29]. The expression fragment was constructed as Kozak sequence—signal peptide: MGWSCIILFLVATATGVHS—Gn D20–C447—His tag—stop codon for Gn and MGWSCIILFLVATATGVHS—Gc C563~N1026—His tag—stop codon for Gc. The DNA sequences were synthesized by Y-Clone Ltd. (Suzhou, China), and were cloned into pVAX1 plasmid to generate pVAX1-GnRFC or pVAX1-GcRFC expression vector. We transfected 293F or CHO cells and purified Gn and Gc, respectively, by Ni+NTA beads (Smart-Lifesciences, Suzhou, China) following the manufacture’s protocol. Gn and Gc were identified by western blot analysis, and anti-His antibody (Santa Cruz, USA) served as the primary antibody at a dilution of 1:300.
3
Purification of Bacterial Fusion Proteins
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To express ZapE and K72A, the expression vector pET28a was used. The zapE and zapEK72A each were amplified from PAO1 genomic DNA and pUCP20‐K72A with 28a‐zapE‐F and 28a‐zapE‐R. For PqsH, the expression vector pMAL‐C5x was chosen, and the pqsH was amplified with MAL‐pqsH‐F and MAL‐pqsH‐R. Gibson Assembly Master Mix (NEB) was applied to fuse the amplicons with the expression vectors. The fusion gene constructs were then transformed into E. coli strain BL21 (DE3). HIS‐ZapE and HIS‐K72A were purified on Ni NTA beads (Smart Lifesciences) independently
19 (link), and MBP‐PqsH was purified on Dextrin Beads 6FF (Smart Lifesciences) as previously reported
13 (link). The fusion proteins were further purified by ion exchange on a Mono Q 5/50 GL column (GE Biosciences) and then followed by a gel filtration on a HiLoad 16/60 Superdex 200 size exclusion column (GE Biosciences).
19 (link), and MBP‐PqsH was purified on Dextrin Beads 6FF (Smart Lifesciences) as previously reported
13 (link). The fusion proteins were further purified by ion exchange on a Mono Q 5/50 GL column (GE Biosciences) and then followed by a gel filtration on a HiLoad 16/60 Superdex 200 size exclusion column (GE Biosciences).
4
Recombinant Protein Purification and EMSA
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ZmHsf28 was subcloned into pET28a for recombinant expression in the Escherichia coli BL21(DE3) strain. The recombinant protein was purified using the Ni-NTA beads (Smart-Lifesciences, Changzhou, China). Promoter fragments of target genes containing specific HSE motifs were synthesized with biotin labeling (Sango, Shanghai, China). Unlabeled probes were used for competition. EMSA was conducted with a chemiluminescence EMSA kit (Beyotime, Shanghai, China), as described previously [85 (link)]. Probes were listed in Table S4 .
5
Recombinant Protein Expression and Purification
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Recombinant proteins were expressed in E. coli strain BL21 (DE3) cultured in LB medium, and induced with 0.1-1 mM IPTG at 18 C for 16 hr. Cells were collected by centrifugation and resuspended in PBS containing 0.5% Tween and 1 mM PMSF, followed by ultrasonication. GST-eEF2 was purified using glutathione beads (Smart-Lifesciences) and eluted by 15mM glutathione. AMPKg1-His, AMPKg3-His and MBP-PPP6C-His were purified using Ni NTA beads (Smart-Lifesciences), and eluted by 250 mM imidazole. MBP-PPP6C, MBP-AMPKg1-CBS3, and MBP-AMPKg1-CBS3-4 were purified using dextrin beads (Smart-Lifesciences) and eluted by 10 mM maltose monohydrate. Eluates were then subjected to dialysis.
For SPR analysis, AMPKb1 was co-expressed with AMPKg1-His in E. coli. 1 mM ATP or 300 mM AMP were added during purification, and AMPKb1/AMPKg1-His complex was further purified by a Superdex 200 10/300 GL size-exclusion column (Cytivalifesciences). 83His-PPP6C bacmid was prepared from the Bac-to-Bac Baculovirus expression system (Thermo Fisher). SF9 cells in SIM serum-free medium (Sinobiological) at a density of 2310 6 cells/ml were infected with recombinant virus and incubated at 27 C for 72 hr. His-tag purification was performed as above, and PPP6C monomers were separated from aggregates by a Superdex 200 10/300 GL size-exclusion column.
For SPR analysis, AMPKb1 was co-expressed with AMPKg1-His in E. coli. 1 mM ATP or 300 mM AMP were added during purification, and AMPKb1/AMPKg1-His complex was further purified by a Superdex 200 10/300 GL size-exclusion column (Cytivalifesciences). 83His-PPP6C bacmid was prepared from the Bac-to-Bac Baculovirus expression system (Thermo Fisher). SF9 cells in SIM serum-free medium (Sinobiological) at a density of 2310 6 cells/ml were infected with recombinant virus and incubated at 27 C for 72 hr. His-tag purification was performed as above, and PPP6C monomers were separated from aggregates by a Superdex 200 10/300 GL size-exclusion column.
6
Recombinant Protein Expression and Purification
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The genes encoding A1L35HR2m, HR2mL35A1, A1L5HR2m and A1HR2m were synthesized and cloned into a pET28a vector by Genewiz, Suzhou, China. The genes encoding A3L35HR2m, A7L35HR2m and A1L35HR2mdCdN were generated by using overlap PCR and cloned into a pET28a. pET28a-A1L35HR2m, pET28a-HR2mL35A1, pET28a-A1L5HR2m, pET28a-A1HR2m, pET28a-A3L35HR2m, pET28a-A7L35HR2m and pET28a-A1L35HR2mdCdN plasmids were transformed separately into E. coli BL21 (DE3). The cells were shaken at 37°C in LB medium until the OD600 reached 0.8. Protein expression was induced with 0.5 mM isopropyl 1-thio-β-D-galactopyranoside for 6 h at 37°C. Then, the cells were harvested, sonicated in PBS with 1% Triton and centrifuged at 1,2000 × g for 10 min. The supernatant was loaded onto Ni-NTA beads (Smart-Lifesciences, Cat. SA004100) and washed with 10 mM imidazole in PBS. Proteins were then eluted with elution buffer (250 mM imidazole in PBS). The peak fractions were collected and concentrated with a 3 kDa ultrafiltration tube (Millipore, Germany). The proteins were then analyzed by SDS‒PAGE and LC‒MS.
7
Recombinant Anti-Fab Nanobody Expression and Purification
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The DNA sequence of anti-Fab Nb was cloned into pET24 (+) vector which bears an N-terminal PelB signal sequence55 (link) and a C-terminal 6× His-tag. The Nb would be expressed and translocated to the periplasm of Escherichia coli strain BL21. The bacteria were cultured in LB medium at 37 °C until OD600 reached about 0.8. Then the protein expression was induced by addition of 1 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG) for 6 h.
For Nb purification, the cell pellets were lysed by sonication in lysis buffer. Cell debris was removed by centrifuging at 18,000 rpm for 1 h. Next, the supernatant was mixed with pre-equilibrated Ni-NTA beads (Smart-Lifesciences) and incubated at 4 °C for 1 h. The beads were sequentially washed with 20 column volumes of Buffer B containing 25 mM and 50 mM imidazole, and then the target protein was eluted with 3 column volumes of Buffer C (25 mM HEPES pH 7.5, 150 mM NaCl, and 300 mM imidazole). The protein was further purified by SEC using a Superdex 200 Increase 10/300 column (GE Healthcare) equilibrated with Buffer B. The protein samples of the peak fractions were collected and concentrated to 5 mg/mL.
For Nb purification, the cell pellets were lysed by sonication in lysis buffer. Cell debris was removed by centrifuging at 18,000 rpm for 1 h. Next, the supernatant was mixed with pre-equilibrated Ni-NTA beads (Smart-Lifesciences) and incubated at 4 °C for 1 h. The beads were sequentially washed with 20 column volumes of Buffer B containing 25 mM and 50 mM imidazole, and then the target protein was eluted with 3 column volumes of Buffer C (25 mM HEPES pH 7.5, 150 mM NaCl, and 300 mM imidazole). The protein was further purified by SEC using a Superdex 200 Increase 10/300 column (GE Healthcare) equilibrated with Buffer B. The protein samples of the peak fractions were collected and concentrated to 5 mg/mL.
8
Purification of PW-pGH28-3 Protein from BL21 (DE3)
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The BL21 (DE3) strain harboring PW-pGH28-3 was incubated overnight in a shaker at 37 °C and 200 rpm. Then, 4 mL of the overnight culture was inoculated to 200 mL fresh LB medium (50 µg/mL kanamycin) in 1 L shake flask. When the OD600 reached 0.6–0.8, IPTG was added to reach 100 µM, and the cells were cultivated at 25 °C for another 6 h. Cells were harvested and washed three times with PBS, then suspended in 20 mL PBS with 1 mM phenylmethylsulfonyl fluoride (PMSF, protease inhibitor). The cells were disrupted by sonication. The cell lysates were centrifuged at 12,000 rpm at 4 °C for 20 min, and the supernatant was collected as crude enzyme solution. Ni NTA beads (Smart-Lifesciences, Jiangsu, China) were used for protein purification, and the purified PW-pGH28-3 protein was concentrated in 10 kDa ultrafiltration tubes (Merck Millipore, Darmstadt, Germany). The purity of PW-pGH28-3 was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the concentration was measured with the Bradford method (Sangon Biotech, Shanghai, China).
9
Aptamer Selection against Mouse IDO1
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At the beginning of the screening process, 50 μg recombinant His-tagged mouse IDO1 protein was immobilized to Ni-NTA beads (Smart-Lifesciences). Then the beads were incubated with 5 nmol ssDNA library in binding buffer (10 mM Tris, 150 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1 mM CaCl2, pH 7.5), supplemented with 1 mg/mL BSA and 100 μg/L tRNA, for 1 h at 4°C. In round #4, bare Ni-NTA beads were introduced as negative selection. After washing with washing buffer (10 mM Tris, 150 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 0.05% (v/v) Tween 20, pH 7.5), sequences bound to the target protein were retrieved in ddH2O by heating to 95°C for 10 min. The supernatant was collected and subjected to PCR to amplify target binding sequences. Next, 5' FAM-labeled forward primer and 5' biotin-labeled reverse primer were used for PCR amplification, and the PCR products were incubated with streptavidin sepharose beads (GE Healthcare Life Sciences) for 30 min at 37°C. NaOH in the concentration of 200 mM was used to separate double-stranded chains of PCR products, and the eluted 5' FAM-labeled amplicons were neutralized by HCl. Then the ssDNA amplicons pool was put into the next SELEX cycle, and the enrichment of candidates in each round was monitored by flow cytometric analysis. Enriched DNA pools from round #13 were cloned into TA vectors and sequenced.
10
Recombinant Foldon-Fused HR1 Peptides
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HR1SFd, HR1MFd, and HR1LFd peptides were obtained by fusing the T4 bacteriophage fibritin-derived foldon peptide directly to the C terminus of HR1-derived short (HR1S), medium (HR1M) and long (HR1L) peptides, respectively (Fig. 1C ). The genes of HR1SFd, HR1MFd, and HR1LFd were synthesized and cloned into a pET28a vector by Genewiz, Suzhou, China.
pET28a-HR1SFd, pET28a-HR1MFd, and pET28a-HR1LFd plasmids were transformed intoE. coli BL21(DE3), respectively. The cells were incubated at 37°C in an LB medium until the OD600 reached 0.8. The peptides were induced with 0.5 mM isopropyl 1-thio-β-d -galactopyranoside for 6 h at 37°C. Then the cells were harvested, sonicated in PBS with 1% Triton and centrifuged at 1,2000 g for 10 min. The supernatant was loaded into Ni-NTA beads (Smart-Lifesciences, catalog no. SA004100) and washed with 10 mM imidazole in PBS. Peptides were then eluted with elution buffer (250 mM imidazole in PBS). The peak fractions were collected and concentrated with a 10 kDa ultrafiltration tube (Millipore, Germany). The peptides were then analyzed by size exclusion chromatography (Superdex 75 Increase 10/300 GL, GE Healthcare) in the CH3COONa/CH3COOH buffer (pH 6.0).
pET28a-HR1SFd, pET28a-HR1MFd, and pET28a-HR1LFd plasmids were transformed into
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