The largest database of trusted experimental protocols

Ni sepharose 6 fast flow resin

Manufactured by GE Healthcare
Sourced in United States, Sweden, United Kingdom

Ni Sepharose 6 Fast Flow resin is a nickel-charged agarose-based affinity chromatography resin designed for the purification of histidine-tagged proteins. The resin features a porous structure that provides a large surface area for protein binding and fast flow rates, enabling efficient and high-capacity protein purification.

Automatically generated - may contain errors

46 protocols using ni sepharose 6 fast flow resin

1

Recombinant Expression and Purification of TAAs

Check if the same lab product or an alternative is used in the 5 most similar protocols
The cDNA for the TAAs, were sub-cloned into modified pET21b or pET45b vectors to create TAA-BirA-6xHis fusions, as previously described [29 (link)]. TAA fusion vectors plus a fusion tag only control were transformed into BL21(DE3) Escherichia Coli competent cells [29 (link)] and cultured as either 60 ml (Discovery study) or 500 ml (Confirmation study) volumes of TB overnight express auto-induction medium (Novagen). For NY-ESO-1, EpCAM, VIM and Annexin II, 500 ml cultures were used for the Discovery study instead of 60 ml. Proteins were purified as previously described using either Ni Sepharose 6 Fast Flow resin (GE Healthcare) in 24 well filter plates [29 (link)] (Discovery study) or 5 ml HisTrap FF crude prepacked columns (GE healthcare) [30 (link)] (Confirmation study). Proteins were analyzed for molecular weight and purity using SDS-PAGE and Western Blotting and quantified by Bradford assay (Biorad).
+ Open protocol
+ Expand
2

Recombinant Protein Purification in E. coli

Check if the same lab product or an alternative is used in the 5 most similar protocols
E. coli C321.ΔA strain harboring plasmid pBK-BocLysRS and a pGFPUV-NCUA variant was cultured in 5 mL LB media containing kanamycin and chloramphenicol at 30 °C. After 18 hours of cultivation, 0.5 mL culture was sub-cultured in 50 mL medium. The protein expression was induced at the OD600nm of 0.6 by additions of IPTG (0.5 mM) and Boc-Lys (5 mM). Cells were collected by centrifugation at 5 000 g and 4 °C for 15 min. Harvested cells were resuspended in lysis buffer containing potassium phosphate (20 mM, pH 7.4), NaCl (150 mM) and imidazole (10 mM). Cells were subsequently disrupted by sonication. Cellular debris was removed by centrifugation (21 000 g, 30 min, 4 °C). The cell-free lysate was applied to Ni Sepharose 6 Fast Flow resin (GE Healthcare). Protein purification followed manufacture's instructions. Protein concentrations were determined by Bradford assay (Bio-Rad). Purified protein was isolated by SDS-PAGE and digested with trypsin prior to MS analysis.
+ Open protocol
+ Expand
3

Recombinant Protein Expression and Purification

Check if the same lab product or an alternative is used in the 5 most similar protocols
The two recombinant plasmids were used to transform Rosetta™ (DE3) pLysS E. coli cells. For recombinant protein production, 100 mL of auto-induction medium containing kanamycin (50 μg/mL) and chloramphenicol (34 μg/mL) was inoculated (1/100 v/v) for each protein. Cultures were then incubated for 24 h at 25 °C in a Multitron Standard shaking incubator (INFORS-HT, Switzerland) (300 rpm). Cells were collected by centrifugation and resuspended in a volume of lysis buffer dependent on the final culture OD of the individual expression, which was then frozen at −80 °C overnight. Cells were thawed, and incubated with 10 µg/mL DNAse and 20 mM MgSO4 for 30 min shaking at 24 °C. To ensure complete cell lysis, cells were sonicated (Soniprep 150, MSE, UK) for 5 min (power 15, 30 s ON/OFF cycles), and subsequently centrifuged for 30 min at 20,000× g. Proteins were then purified using Ni Sepharose 6 Fast Flow resin (GE Healthcare, Uppsala, Sweden) and eluted with 250 mM imidazole elution buffer (50 mM Tris, 300 mM NaCl, 250 mM imidazole, pH 8.0). Purified protein was dialyzed overnight against protein buffer (50 mM Tris, 300 mM NaCl, pH 8.0) in Thermo Scientific™ Slide-A-Lyzer™ 10K MWCO dialysis cups, prior to storage at −80 °C until further use.
+ Open protocol
+ Expand
4

Stoichiometric Protein Labeling Protocol

Check if the same lab product or an alternative is used in the 5 most similar protocols
Proteins were labelled according to an established protocol33 (link),74 (link). The cysteines were stochastically labelled with the maleimide derivative of the dyes TMR, Alexa Fluor 555, Alexa Fluor 647 and Cy5 (ThermoFisher Scientific). His-tagged proteins were incubated in 1 mM DTT to keep all cysteine residues in a reduced state and subsequently immobilized on a Ni Sepharose 6 Fast Flow resin (GE Healthcare). The resin was incubated 2–4 h at 4 °C with 25 nmol of each fluorophore dissolved in 1 ml of labelling buffer 1 (50 mM Tris-HCl pH 7.4, 50 mM KCl, 5% glycerol) and subsequently washed sequentially with 3 ml labelling buffer 1 and buffer 2 (50 mM Tris-HCl pH 7.4, 150 mM KCl, 50% glycerol) to remove unbound fluorophores. Bound proteins were eluted with 500 ml of elution buffer (50 mM Tris-HCl pH 8, 50 mM KCl, 5% glycerol, 500 mM imidazole) The labelled protein was further purified by size-exclusion chromatography (ÄKTA pure, Superdex 75 Increase 10/300 GL, GE Healthcare) to eliminate remaining fluorophores and remove soluble aggregates. For all proteins, labelling efficiencies were higher than 70%, and donor–acceptor pairing at least 20%.
+ Open protocol
+ Expand
5

Periplasmic Expression and Purification of Nanobodies

Check if the same lab product or an alternative is used in the 5 most similar protocols
A series of nanobodies were individually expressed in the periplasm of E. coli strain WK6, which was grown in TB media supplemented with 0.1 mg/ml carbenicillin, 0.1% glucose and 2 mM MgCl2 to an absorbance A600 ~0.7 at 37°C. The culture was induced with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) and incubated in an orbital shaker overnight at 28°C. Cells were harvested by centrifugation, resuspended in TES buffer (200 mM TRIS, pH 8.0; 0.5 mM EDTA; 500 mM sucrose supplemented with 40 mM imidazole) and incubated for 1 hr. To this fraction four times diluted TES buffer was added and incubated for 1 hr. This fraction was cleared by centrifugation at 10,000 g. The supernatant was incubated with Ni Sepharose 6 Fast Flow resin (GE Healthcare) and incubated for 1 hr at room temperature. The beads were washed with buffer containing 20 mM TRIS pH 8.0, 300 mM NaCl and 40 mM imidazole. Protein was eluted with the same buffer supplemented with 300 mM imidazole. The eluted protein was concentrated to less than 1 ml on a 3 kDa cut-off Vivaspin concentrating column (Sartorius) and further purified on a Superdex 75 10/300 GL column (GE Healthcare) equilibrated with 10 mM Na-phosphate (pH 8.0) and 150 mM NaCl. Peak fractions corresponding to nanobody were pooled and spin-concentrated to ~50 mg/ml.
+ Open protocol
+ Expand
6

Production and Purification of SARS-CoV-2 Spike Proteins

Check if the same lab product or an alternative is used in the 5 most similar protocols
Expression plasmids encoding the codon optimized SARS-CoV-2 full length spike and receptor binding domain (RBD) were kindly provided Kizzmekia Corbett and Barney Graham (Vaccine Research Center, Bethesda, USA) and Florian Krammer (Icahn School of Medicine at Mt. Sinai, New York, USA), respectively (Wrapp et al., 2020 (link); Amanat et al., 2020 (link)). Both plasmids were expressed in Freestyle 293-F cells (Thermofisher), maintained in Freestyle 293 Expression Medium (GIBCO/ThermoFisher) at 37°C and 8% CO2 in a humidified incubator shaking at 130 rpm. Cultures totaling 500 mL were transfected with PEI at a density of one million cells per mL. Supernatant was harvested 7 days post transfection, clarified by centrifugation and sterile filtered through a 0.22 μM membrane. The protein was purified using Ni-NTA immobilized metal-affinity chromatography (IMAC) using Ni Sepharose 6 Fast Flow Resin (GE Lifesciences) or NiNTA Agarose (QIAGEN) and gravity flow. After elution the protein was buffer exchanged into 10 mM Tris pH8, 150 mM NaCl buffer before further use or frozen at −80°C for storage.
+ Open protocol
+ Expand
7

SARS-CoV-2 Spike Protein Production

Check if the same lab product or an alternative is used in the 5 most similar protocols
Protein production was performed as described previously (44 (link), 45 (link)). Expression plasmids encoding the codon-optimized SARS-CoV-2 full-length S and RBD were obtained from K. Corbett and B. Graham [Vaccine Research Center, National Institutes of Health (NIH)] (46 (link)) and F. Krammer (Icahn School of Medicine at Mt. Sinai) (47 (link)). Expression was performed in FreeStyle 293-F cells (Thermo Fisher Scientific) maintained in FreeStyle 293 Expression Medium (Gibco) at 37°C and 8% CO2 shaking at 130 rpm. Cultures totaling 500 ml were transfected with polyethylenimine at a density of 1 million cells/ml. Supernatant was harvested 7 days after transfection, clarified by centrifugation, and filtered through a 0.22 μM membrane. The protein was purified using Ni–(nitrilotriacetic acid) NTA immobilized metal-affinity chromatography using Ni Sepharose 6 Fast Flow Resin (GE Lifesciences) or Ni-NTA Agarose (Qiagen) and gravity flow. After elution the protein was buffer exchanged into 10 mM tris (pH 8), 150 mM NaCl buffer (S), or PBS (RBD) and stored at −80°C.
+ Open protocol
+ Expand
8

Expression and Purification of Recombinant Proteins

Check if the same lab product or an alternative is used in the 5 most similar protocols
Proteins were expressed and purified as described previously24 (link),39 (link). Transfections were performed according to the manufacturer’s guidelines (Gibco, Thermo Fisher). Briefly, cells were passaged 1 day before transfection. On the day of transfection, cells were diluted to 3.0 × 106 cells per ml. Desired plasmids were complexed with ExpiFectamine293 and transiently transfected into Expi293F cells. Transfected cells were treated after 16 h with Enhancer 1 and Enhancer 2. Five days posttransfection, culture supernatant was collected, proteins were affinity purified by immobilized metal affinity chromatography using Ni Sepharose 6 Fast flow resin (GE Healthcare) as described previously39 (link). Briefly, the supernatant was twofold diluted with 1× PBS (pH 7.4) bound to a column equilibrated with PBS (pH 7.4). A ten-column volume wash of 1× PBS (pH 7.4), supplemented with 25 mM imidazole was performed. Finally, the bound protein was eluted with a gradient of 200–500 mM imidazole in PBS (pH 7.4). The eluted fractions were pooled and dialyzed three times using a 3–5 kDa (molecular weight cutoff) dialysis membrane (40 mm flat width) (Spectrum Laboratories) against PBS (pH 7.4). Protein concentration was determined by absorbance (A280) using NanoDrop 2000c with the theoretical molar extinction coefficient calculated using the ProtParam tool (ExPASy).
+ Open protocol
+ Expand
9

Tandem Affinity Purification of Proteins

Check if the same lab product or an alternative is used in the 5 most similar protocols
Cells were lysed in “His-purification buffer” [50 mM tris (pH 7.5), 300 mM NaCl, 1.5 mM MgCl2, and 0.15% CA-630 (Sigma-Aldrich)] with the Protease Inhibitor Mix HP (Serva), and the cleared lysate was incubated with a Ni Sepharose 6 Fast Flow resin (GE Healthcare). The unbound material was washed out with His-purification buffer in the Poly-Prep Chromatography Column (Bio-Rad), and proteins were eluted with a “His-elution buffer” [50 mM tris (pH 7.5), 150 mM NaCl, 1.5 mM MgCl2, and 500 mM imidazole]. The eluate was incubated with a StrepTactin Sepharose High Performance resin (GE Healthcare), unbound material was washed out on the Poly-Prep Chromatography Column (Bio-Rad) with “Strep-purification buffer” [50 mM tris (pH 7.5), 300 mM NaCl, and 0.5 mM dithiothreitol (DTT)], and proteins were eluted with “Strep-elution buffer” [50 mM tris (pH 7.5), 150 mM NaCl, 0.5 mM DTT, and 5 mM biotin].
+ Open protocol
+ Expand
10

Recombinant Protein Expression and Purification

Check if the same lab product or an alternative is used in the 5 most similar protocols
The Champion™ pET SUMO expression vector (K30001) and PureLink™ Quick Gel Extraction Kit (K210012) were purchased from Invitrogen. DreamTaq Green PCR Master Mix (K1082), T4 DNA ligase (5U/µl), Rapid DNA Ligation Kit, GeneJET Plasmid Miniprep Kit, isopropyl β-D-1-thiogalactopyranoside (IPTG), and PageRuler™ Unstained Protein ladder were obtained from Thermo Scientific™. Rosetta 2(DE3), Rosetta 2(DE3) pLysS, and Rosetta-gami™ 2(DE3) cells were purchased from Novagen, and HepG2, HCT-116, and MCF-7 cell lines for the biological assay were purchased from ATCC, USA. Terrific broth and tetracycline hydrochloride were purchased from Fisher bioreagents™, USA. Streptomycin sulfate, chloramphenicol, and a purification column (Ni-Sepharose column) were purchased from Sigma. The Ni-Sepharose™ 6 Fast Flow resin was purchased from GE Healthcare Life Sciences. Kanamycin monosulfate was obtained from ICN Biomedicals, Inc. IL28/29 (H-1) sc-365834 mouse monoclonal IgG2a antibody and goat anti-mouse IgG-AP-conjugated antibody were purchased from Santa Cruz Biotechnology. The Rainbow™ Full-range Molecular Marker (Mr 12,000–225,000) was purchased from GE Healthcare Life Sciences. DMEM medium, fetal bovine serum, penicillin–streptomycin and trypsin were purchased from Gibco.
+ Open protocol
+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required

Sign up now

Revolutionizing how scientists
search and build protocols!