Tni cells (Expression Systems) were cultured as suspension cells at 27 °C and 130 rpm in ESF 291 medium (Expression Systems) supplemented with a final 5 µg ml−1 gentamicin (Sigma-Aldrich). The cell density was maintained at 0.5–3.0 × 106 cells ml−1. The U2-OS cells (2-6-3 cell line, a kind gift from David Spector) were cultured as adherent cells at 37 °C, 5% CO2 in DMEM medium supplemented with 2 mM
Tni cells
Manufactured by Expression Systems
Tni cells are a type of insect cell line commonly used in the production of recombinant proteins. They are derived from the pupal ovarian tissue of the Trichoplusia ni (Tn) moth species. Tni cells possess the ability to properly fold and post-translationally modify recombinant proteins, making them a valuable tool for the expression of complex eukaryotic proteins.
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Lab products found in correlation
Sf9 cells, by Thermo Fisher Scientific (4 mentions)
Sf 900 2 serum free medium, by Thermo Fisher Scientific (2 mentions)
Esf 921 serum free medium, by Expression Systems (2 mentions)
Gentamicin, by Merck Group (1 mentions)
Sf9 cells, by Expression Systems (1 mentions)
Ni nta, by Qiagen (1 mentions)
Ni nta column, by Takara Bio (1 mentions)
Hace2 avitag protein from 293t cells, by ACROBiosystems (1 mentions)
Bac to bac baculovirus expression system, by Thermo Fisher Scientific (1 mentions)
Pfastbac1 expression vector, by Thermo Fisher Scientific (1 mentions)
7 protocols using tni cells
1
Culturing Tni and U2-OS Cell Lines
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Tni cells (Expression Systems) were cultured as suspension cells at 27 °C and 130 rpm in ESF 291 medium (Expression Systems) supplemented with a final 5 µg ml−1 gentamicin (Sigma-Aldrich). The cell density was maintained at 0.5–3.0 × 106 cells ml−1. The U2-OS cells (2-6-3 cell line, a kind gift from David Spector) were cultured as adherent cells at 37 °C, 5% CO2 in DMEM medium supplemented with 2 mMl -glutamine, 1% penicillin/streptomycin, 100 µg ml−1 hygromycin B (for LacO array maintenance), and 10% fetal bovine serum.
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Tni cells (Expression Systems) were cultured as suspension cells at 27 °C and 130 rpm in ESF 291 medium (Expression Systems) supplemented with a final 5 µg ml−1 gentamicin (Sigma-Aldrich). The cell density was maintained at 0.5–3.0 × 106 cells ml−1. The U2-OS cells (2-6-3 cell line, a kind gift from David Spector) were cultured as adherent cells at 37 °C, 5% CO2 in DMEM medium supplemented with 2 mM
2
Insect cell line-based protein expression
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Standard, commercial insect cell lines used this study include Sf9 cells (Invitrogen) and Tni (High-Five) cells (Expression System). Sf9 cells were used for baculovirus preparation and maintained in Sf900 II serum free medium (Thermo Fisher). Tni cells were used for large-scale protein expression and are maintained in ESF 921 serum free medium (Expression Systems).
3
SARS-CoV-2 Spike Protein Production
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The SARS-CoV-2 spike protein (GenBank: QHD43416.1) was codon optimized and modified to remove the transmembrane domain region. It was synthesized on a pABbee™-FH plasmid by GenScript (USA). Recombinant baculovirus was generated by transfecting the spike plasmids into Sf9 cells (Expression Systems) using the ProFold™-ER1 system (AB vector). Clones were screened following plaque purification, and master seed stocks were prepared, characterized for identity, and used to prepare working virus stocks. The working stocks were subcultured on Sf9 cells at 27 °C with shaking at 130 rpm for 120 h and were subsequently used to infect suspension cultures of TNI cells (Expression Systems) at an MOI of 5–10. The infected TNI cells were incubated at 27 °C with shaking at 130 rpm for 72 h for protein expression. The spike protein was purified by nickel-nitrilotriacetic acid (Ni-NTA) (Qiagen, Toronto, Canada), and the final product was confirmed to be functional by evaluation of the antigen using an in-house developed ELISA with positive and negative SARS-CoV-2 sera.
4
Recombinant Tubulin Protein Purification
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Human αβ-tubulin (non-tagged TUBA1B gene and TUBB3
gene with a cleavable His-tag at the C-terminus) was expressed in
insect cells as described previously.23 (link) Briefly, Tni cells (Expression Systems) and ESF-921 insect cell
medium (Expression Systems) were used for expression. Cells were harvested
approximately ∼42 h post-infection, re-suspended in 3 volumes
of lysis buffer (25 mM Hepes, pH 7.4, 30 mM imidazole, 1 mM MgSO4, 50 μM GTP), and lysed using a glass dounce. Lysate
was clarified by centrifugation, and recombinant tubulin was purified
by Ni-affinity (5 mL Ni-NTA column, TaKaRA) and anion exchange (4
mL Source-Q column, GE Amersham) chromatography. The His-tag was removed
by TEV protease (2 h on ice using a TEV at 0.2 mg/mL final concentration)
prior to anion exchange chromatography. Peak fractions were pooled,
concentrated to 15–20 μM, buffer-exchanged to BRB80 with
50 μM GTP, flash-frozen on liquid nitrogen in 100 μL aliquots,
and stored at −80 °C.
gene with a cleavable His-tag at the C-terminus) was expressed in
insect cells as described previously.23 (link) Briefly, Tni cells (Expression Systems) and ESF-921 insect cell
medium (Expression Systems) were used for expression. Cells were harvested
approximately ∼42 h post-infection, re-suspended in 3 volumes
of lysis buffer (25 mM Hepes, pH 7.4, 30 mM imidazole, 1 mM MgSO4, 50 μM GTP), and lysed using a glass dounce. Lysate
was clarified by centrifugation, and recombinant tubulin was purified
by Ni-affinity (5 mL Ni-NTA column, TaKaRA) and anion exchange (4
mL Source-Q column, GE Amersham) chromatography. The His-tag was removed
by TEV protease (2 h on ice using a TEV at 0.2 mg/mL final concentration)
prior to anion exchange chromatography. Peak fractions were pooled,
concentrated to 15–20 μM, buffer-exchanged to BRB80 with
50 μM GTP, flash-frozen on liquid nitrogen in 100 μL aliquots,
and stored at −80 °C.
5
Quantifying SARS-CoV-2 Spike Protein Binding
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A sensor chip was captured with recombinant hACE2‐AviTag protein from 293T cells (Acro Biosystems, Newark, DE) followed by injection of 300 μl of freshly prepared serial dilutions of S‐Ecto‐HexaPro(+F), S‐Ecto‐HexaPro(‐F), S‐RBD‐eGFP, and S‐Ecto‐eGFP made with Tni cells (Expression Systems) and passed over it at a flow rate of 50 μl/min (contact duration 180 s) for the association, and disassociation was performed over a 600‐s interval. A mock surface and buffer‐only injections were used to correct for background signal. Bio‐Rad ProteOn Manager (version 3.1) was used for data processing.
6
Recombinant Telomere Protein Expression
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Human TRF1 (NP_059523.2), TRF2 (XP_005256180.1), RAP1 (NP_061848.2), TIN2 (NP_036593.2), TPP1 (AAX82621.1), and POT1 (NP_056265.2) cDNAs were individually cloned into the pFastBac1 expression vector (Invitrogen). The TRF1, TRF2, and RAP1 constructs have N-terminal hexa-histidine (6XHis) tags while TIN2, TPP1, and POT1 have PreScission-cleavable N-terminal 6XHis-MBP tags. The expression vectors were used to make recombinant baculoviruses based on the protocol established in the Bac-to-Bac Baculovirus Expression System (Invitrogen). The baculovirus stocks were titered by gp64 detection using flow cytometry analysis (Expression Systems). Yield-optimized amounts of baculoviruses were used to infect 2–4 liters of Tni cells (Expression Systems) at a cell density of 1.5–2.0 × 106 cells ml−1. The infected cells were then incubated in a shaker for 66–72 h (27 °C, 130 rpm) before they were collected. The collected cells were either immediately used for protein purification or snap-frozen using liquid nitrogen for later purification.
7
Insect cell line-based protein expression
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Standard, commercial insect cell lines used this study include Sf9 cells (Invitrogen) and Tni (High-Five) cells (Expression System). Sf9 cells were used for baculovirus preparation and maintained in Sf900 II serum free medium (Thermo Fisher). Tni cells were used for large-scale protein expression and are maintained in ESF 921 serum free medium (Expression Systems).
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