Genes were cloned into pBAD, pETDUET1, and pET22b vectors for expression with either His or 3V5 tags. Cultures were grown in 10 ml LB with appropriate antibiotics to OD600 of 0.6, and induced with 0.1% [w/v] l -arabinose at 30 °C for 3 h or 1 mM IPTG overnight at 20 °C. Cells were centrifuged at 4500 r.p.m. for 10 min and resuspended in 1 ml of lysis buffer (20 mM Tris pH 8.0, 500 mM NaCl, 50 mM imidazole with protease inhibitor (Thermo Scientific)). Resuspended cells were sonicated (20 × 5 s) and cell debris was removed by centrifugation (15,000 × g for 15 min). Clarified supernatants were mixed and loaded to Ni-NTA resin (Smart-lifesciences), washed five times with wash buffer (20 mM Tris pH 8.0, 500 mM NaCl, 50 mM imidazole), and eluted in 100 µl elution buffer (20 mM Tris pH 8.0, 500 mM NaCl, 500 mM imidazole). Cell lysates and eluted samples were analyzed by western blotting.
Ni nta resin
Manufactured by Smart-Lifesciences
Sourced in China
Ni-NTA resin is a chromatography medium used for the purification of recombinant proteins with a histidine-tag. It consists of nickel-nitrilotriacetic acid (Ni-NTA) immobilized on agarose beads. The nickel ions present in the resin bind to the histidine residues on the target protein, allowing it to be captured and separated from other components in the sample.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor, by Thermo Fisher Scientific (3 mentions)
Gst resin, by Smart-Lifesciences (3 mentions)
Ez link sulfo nhs lc lc biotin, by Thermo Fisher Scientific (2 mentions)
96 well black microplate, by Greiner (2 mentions)
Multiskan fc, by Thermo Fisher Scientific (2 mentions)
Streptavidin, by Sartorius (1 mentions)
Bi 224436, by MedChemExpress (1 mentions)
Mouse anti gst antibody, by Merck Group (1 mentions)
Pierce tmb substrate kit, by Thermo Fisher Scientific (1 mentions)
Anti human igg hrp, by Jackson ImmunoResearch (1 mentions)
Polyethylenimine (pei), by Polysciences (1 mentions)
Sorvall st8, by Thermo Fisher Scientific (1 mentions)
Halt protease inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Peg8000, by Sangon (1 mentions)
Palmitate, by Merck Group (1 mentions)
Recombinant human insulin, by Novo Nordisk (1 mentions)
White 384 shallow well microplate, by PerkinElmer (1 mentions)
Anti 6his xl665, by PerkinElmer (1 mentions)
Streptavidin sa biosensor, by Molecular Devices (1 mentions)
12 protocols using ni nta resin
1
Recombinant Protein Expression and Purification
2
Purification of His-tagged Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Genes of interest were cloned into pET and pBAD vectors with His, 3V5 or FLAG epitope tags and expression was induced in E. coli individually. Cells were grown in LB with appropriate antibiotics to OD600 of 0.6–0.8, and induced with 1 mM IPTG for 18 h at 20°C for pET vectors and with 0.1% arabinose for 3 h at 30°C for pBAD vectors. Pellets were collected by centrifugation, resuspended in lysis buffer (20 mM Tris, 500 mM NaCl, 50 mM imidazole, pH 8.0 with protease inhibitor (Thermo Scientific)), and lysed by sonication. After centrifugation to remove cell debris, supernatants were mixed as input samples. Samples were loaded to Ni-NTA resin (Smart-lifesciences), then washed 4–5 times with wash buffer (20 mM Tris pH 8.0, 500 mM NaCl, 50 mM imidazole), and eluted in elution buffer (20 mM Tris pH 8.0, 500 mM NaCl, 500 mM imidazole). Input and elution samples were analyzed by Western blot. Expression of V5-tagged TseP and FLAG-tagged TseI was unstable and excluded in the pull-down analysis.
3
High-Throughput Protein Interaction Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Anti-6His-XL665 Cryptate antibodies and anti-GST-Eu Cryptate antibodies, white 384-shallow well microplate were purchased from PerkinElmer. 96-well Black microplates were purchased from Greiner Bio-One. Streptavidin (SA) biosensors were purchased from Sartorius ForteBio. EZ-Link™ Sulfo-NHS-LC-LC-Biotin was purchased from Thermo Fisher Scientific. BI224436 was purchased from MedChem Express (Shanghai, China). Compounds were obtained from National Compound Resource Centre (Shanghai, China) and stock solutions (10 mM) were stored protected from light at −80 °C. Ni-NTA resin and GST resin were purchased from Smart-Lifesciences (Changzhou, China). All general biochemical reagents were obtained from AMRESCO (Solon, USA).
4
Recombinant Protein Expression and Purification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Genes of interest were cloned into pET and pBBRT vectors for expression. Cells were grown in liquid LB medium with appropriate antibiotics to exponential phase (OD600 ~ 0.6) at 37°C and induced with 1 mM IPTG overnight at 20°C for pET vectors or 100 ng/ml anhydrotetracycline (aTc) for 3 h at 37°C for pBBRT vectors. Cells were harvested by centrifugation at 4500g for 10 min, resuspended in lysis buffer (20 mM Tris, pH 8.0, 500 mM NaCl, 50 mM imidazole with protease inhibitor [Thermo Scientific]), and lysed by sonication. After cell debris was removed by centrifugation at 15,000g for 5 min, supernatants were mixed and incubated with Ni‐NTA resin (Smart‐lifesciences) at 4°C for 1 h. The samples were then washed five times with wash buffer (20 mM Tris, pH 8.0, 500 mM NaCl, 50 mM imidazole), and eluted in elution buffer (20 mM Tris, pH 8.0, 500 mM NaCl, 500 mM imidazole). Input and eluted samples were boiled for 10 min prior to SDS‐PAGE and Western blot analysis.
5
RhsB Protein Expression and In Vitro Activity
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RhsB and its variants were expressed using the pETSUMO vectors in E. coli BL21(DE3). Cells were grown in liquid LB medium to exponential phase (OD600 ~ 0.6) at 37°C. For the expression of His‐SUMO‐RhsBCT or its non‐toxic mutants, cells were induced with 1 mM IPTG at 37°C for 5 h. For the expression of His‐SUMO‐RhsBKE‐AA, cells were induced with 1 mM IPTG at 20°C for 18 h. The cells were harvested by centrifugation at 4500g for 10 min. The pellets were resuspended in lysis buffer (20 mM Tris‐HCl, 150 mM NaCl, 10 mM imidazole, pH 8.0) and lysed by sonication. Lysates were centrifuged at 15,000g for 20 min and the supernatants were incubated with Ni‐NTA resin (Smart‐lifesciences). Proteins were eluted in elution buffer (20 mM Tris‐HCl, 150 mM NaCl, and variable concentrations of imidazole, pH 8.0). Eluted samples were analyzed by SDS‐PAGE analysis.
Protein activity in vitro was detected by incubating with 100 ng plasmid at 37°C for 1 h. NEB CutSmart buffer (50 mM potassium acetate, 20 mM tris‐acetate, 10 mM magnesium acetate, 100 µg/ml bovine serum albumin, pH 7.9) was chosen as the reaction buffer. Purified proteins (0.1 μg) and 0.5 units DNase I (positive control) were used separately in each reaction.
Protein activity in vitro was detected by incubating with 100 ng plasmid at 37°C for 1 h. NEB CutSmart buffer (50 mM potassium acetate, 20 mM tris‐acetate, 10 mM magnesium acetate, 100 µg/ml bovine serum albumin, pH 7.9) was chosen as the reaction buffer. Purified proteins (0.1 μg) and 0.5 units DNase I (positive control) were used separately in each reaction.
6
Purification and Characterization of Recombinant Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All general biochemical reagents were obtained from AMRESCO (Solon, USA). Ni-NTA resin and GST resin were purchased from Smart-Lifesciences (Changzhou, China). Ni-NTA Magnetic Agarose Beads were purchased from BEAVER Nano-technologies (Suzhou, China). 96-well nonbinding surface (NBS) microplates were purchased from Corning (New York, USA). Rabbit anti-mouse IgG alkaline phosphatase conjugated antibody and mouse anti-GST antibody were purchased from Sigma (St. Louis, USA). INH5 was synthesized by GL Biochem (Shanghai, China).
7
Purification and Characterization of THADA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
THADA-His vector was purchased from General Biosystem and constructed as described above. Proteins were expressed individually in Transetta (DE3) cells grown in LB medium with shaking at 220 rpm, 37°C. Overproduction of the protein was then induced by isopropylthio-β-galactoside (IPTG) to a final concentration of 0.2 mM at log phase. After grown for an additional 14 hours at 30 °C, the cells were harvested by centrifugation at 6000× g for 10 min. The bacterial cells were lysed via high pressure homogenizer in buffer containing 20 mM Tris-HCl (pH 8.0), 300 mM NaCl, 1 mM PMSF, 5 µg/mL DNase, 2 mM MgCl2. The THADA protein was purified on Ni-NTA resin (Smart Life Sciences) followed by the application of anion exchange column (HiTrap Capto Q ImpRes, 5 mL) and size exclusion chromatography (Superdex 200 Increase 10/300 GL). Purified protein was concentrated and exchanged into PBS and stored at −80°C.
For ELISA, 2–16 µg/mL of THADA was added to a 96-well ELISA plate and incubated overnight at 4°C. Following five washes with PBST, non-specific binding sites were blocked with 2% BSA in PBS; 1 µg/mL PD-L1-Fc was added to the wells followed by antihuman IgG HRP (Jackson Immuno Research). Enzyme detection was achieved using Pierce TMB Substrate Kit (Thermo Scientific) and signal intensities were quantified using the Thermo Scientific Multiskan FC.
For ELISA, 2–16 µg/mL of THADA was added to a 96-well ELISA plate and incubated overnight at 4°C. Following five washes with PBST, non-specific binding sites were blocked with 2% BSA in PBS; 1 µg/mL PD-L1-Fc was added to the wells followed by antihuman IgG HRP (Jackson Immuno Research). Enzyme detection was achieved using Pierce TMB Substrate Kit (Thermo Scientific) and signal intensities were quantified using the Thermo Scientific Multiskan FC.
8
Stabilized SARS-CoV-2 S Trimer Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The constructs used for expression of stabilized soluble MERS-CoV, SARS-CoV, SARS-CoV-2, and Omicron BA.1 S2P S trimer proteins were obtained from our previous studies47 (link). The mammalian expression plasmid for dimeric soluble ACE2 was purchased from Addgene. SARS-CoV-2 NTD (aa 1–305) and RBD (aa 319–541) were separately cloned into mammalian expression vector pCMV3 with an 8× His tag and 2× Strep-tag II tags at the C terminus. Expi293 cells were used for transient transfection with the suitable S2P-stabilized S-expression plasmids or other vectors by using 1 mg/mL polyethylenimine (PEI, Polysciences). The supernatant was harvested and the S trimer was purified using Ni-NTA resin from Smart Lifesciences (Changzhou, China, Ni Smart Beads 6FF: SA036100) in accordance with the manufacturer’s protocol five days after transfection. Prior to use, all proteins were further evaluated for size and purity through SDS-PAGE.
9
Recombinant Protein Expression and Purification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The tree TLP genes were expressed in the E. coli BL21 (DE3) strain. The cells grown at 37 °C with shaking at 180 rpm in 250 mL of LB broth, containing 50 μg/mL Ampicillin until an OD600 reached 0.8. The expression of the recombinant protein was induced by the addition of isopropyl β-D-1-thiogalactopyranoside (IPTG) at the final concentration of 0.1 mmol/L, followed by cultivation for 24 h at 15 °C with 160 rpm (High speed tabletop centrifuge, Sorvall ST8, Thermo Fisher Scientific Corporation, MA, USA). The culture was harvested by centrifugation (10,000×g for 15 min at 4 °C), and the cell pellets were re-suspended in 30 mL of buffer A (50 mmol/L Tris–HCl pH 8 and 100 mmol/L NaCl) and were incubated for 30 min on ice. The cells were disrupted by sonication, and the insoluble fraction was collected by centrifugation (14,400 rpm for 60 min at 4 °C).
The three protein purification was performed in a single gravity flow chromatography step using a column packed with 1 mL of Ni-NTA resin (smart-lifesciences, China) and was equilibrated with buffer A. The purified protein was eluted using a five-step gradient of imidazole (10, 20, 80 and 300 mmol/L) in buffer A, each step containing 10 mL of the respective buffer. The purity of the protein samples was estimated by SDS-PAGE and stained with Coomassie brilliant blue G-250.
The three protein purification was performed in a single gravity flow chromatography step using a column packed with 1 mL of Ni-NTA resin (smart-lifesciences, China) and was equilibrated with buffer A. The purified protein was eluted using a five-step gradient of imidazole (10, 20, 80 and 300 mmol/L) in buffer A, each step containing 10 mL of the respective buffer. The purity of the protein samples was estimated by SDS-PAGE and stained with Coomassie brilliant blue G-250.
10
Affinity-based Protein Interaction Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
His-tagged bait and V5-tagged prey genes were cloned into pETDuet and pBAD vectors. His-tagged TssAN (E2-Q126) was prone to precipitation when expressed, thus a His-tagged maltose binding protein (MBP)-TssAN with increased solubility was used instead. E. coli BL21 DE3 carrying different pETDuet plasmids or E. coli T-Fast with different pBAD plasmids were grown in 10 ml LB media (1% [wt/vol] tryptone, 0.5% [wt/vol] yeast extract, 0.5% [wt/vol] NaCl) with appropriate antibiotic at 37 °C to an OD600 of approximately 0.6. Cells were induced with 1 mM IPTG at 20 °C for 16 h or with 0.1% [wt/vol] L-arabinose at 30 °C for 3 h. Cells were then harvested and resuspended in 1 ml of lysis buffer (20 mM Tris (pH 8.0), 500 mM NaCl and 50 mM imidazole with 1× Halt protease inhibitor cocktail (Thermo Scientific)). After sonication, the lysates were collected by centrifugation (13,800 × g for 10 min). Lysate and Ni-NTA resin (Smart lifesciences) were mixed and incubated at 4 °C for 1 h, washed with 1 ml wash buffer (20 mM Tris (pH 8.0), 500 mM NaCl and 50 mM imidazole), eluted in 100 μl elution buffer (20 mM Tris (pH 8.0), 500 mM NaCl and 500 mM imidazole) and analyzed by western blot.
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
Revolutionizing how scientists
search and build protocols!