ESF 921 media

Manufactured by Advancion

Sourced in United States

The ESF 921 media is a laboratory equipment product designed for cell culture applications. It serves as a growth medium to support the cultivation and maintenance of various types of cells. The core function of the ESF 921 media is to provide the necessary nutrients, growth factors, and a suitable environment for cell proliferation and survival.

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Lab products found in correlation

44 protocols using ESF 921 media

Recombinant expression and purification of human peroxisomal acyl-CoA oxidase 1

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N-terminal 6xhistidine tagged hPAOX, isoform 1 (Q6QHF9-2; Refseq NP_690875.1) was expressed in baculovirus-infected Sf9 insect cells under the AcMNPV polyhedrin promoter (pPolh) transcriptional control. Codon-optimized cDNA was subcloned into pVL1393 (Expression Systems, 91–012) and co-transfected into Sf9 cells with linearized BestBac™ Baculovirus DNA (Expression Systems 91–002). P2 amplified virus was used to infect Sf9 cells for large-scale production in ESF 921 media (Expression Systems, 96-001) for 57 h to final viability of 85% and cell diameter of 21.3 µm.
6His-hPAOX protein was purified by IMAC. Homogenized lysate (10 ml per 1 g cell paste) in 25 mM HEPES, 500 mM NaCl, 20 mM imidazole, 250U/µl Benzonase (Novagen 71205), and 1x protease cOmplete inhibitor cocktail (Roche 05056489001) was bound to HisPur Ni-NTA resin (ThermoFisher) and eluted stepwise in loading buffer supplemented with 40, 80, 160, and 400 mM imidazole.
Pooled fractions from 10 and 40 mM imidazole elutions were yellow, indicating bound FAD co-factor. Pooled Ni-NTA fractions were concentrated with Jumbosep^TM 10 K MWCO filter (Pall, OD010C65) at 10 °C and loaded onto a Superdex 200 26/600 sizing column (MilliporeSigma) equilibrated with 10 mM HEPES, pH 7.4, 150 mM NaCl. Eluted fractions were pooled and concentrated to 1.47 mg/ml and snap-frozen in liquid nitrogen for long-term storage at −80 °C.

Diaz E., Adhikary S., Tepper A.W., Riley D., Ortiz-Meoz R., Krosky D., Buyck C., Lamenca C.M., Llaveria J., Fang L., Kalin J.H., Klaren V.N., Fahmy S., Shaffer P.L., Kirkpatrick R., Carbajo R.J., Thomsen M, & Impagliazzo A. (2022). Structure of human spermine oxidase in complex with a highly selective allosteric inhibitor. Communications Biology, 5, 787.

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Baculovirus Protein Expression in High Five

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Initial bacmid DNA was generated in DH10Bac cells (Thermo Fisher Scientific) according to manufacturer protocols. Generation of baculovirus was performed in SF9 cells (Thermo Fisher Scientific), SF9 cells were maintained in SF900 iii SFM at 27 C and baculovirus was generated at a cell concentration of ~ 1×10^6 cells/ml. Protein expression was performed in High Five cells (Thermo Fisher Scientific) adapted to and maintained in ESF 921 media (Expression Systems) at 27 C. Viral infections for protein expression were performed at a cell concentration of ~ 2×10^6 cells/ml.

Gestaut D., Zhao Y., Park J., Ma B., Leitner A., Collier M., Pintilie G., Roh S.H., Chiu W, & Frydman J. (2022). Structural visualization of the tubulin folding pathway directed by human chaperonin TRiC/CCT. Cell, 185(25), 4770-4787.e20.

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Insect Cell-Based HBV S1/S2 Protein Expression

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Akshaya Bio Inc. (Edmonton, AB, Canada) kindly provided Sf9 insect cells, which were cultured and maintained in suspension in ESF 921 media (Expression Systems, Davis, CA95618, USA) at 27°C and 110 rpm. All shake flasks were purchased from Fisher Sci. (NH 03842 USA, Cat #: PBV125). In order to express the HBV S1/S2 protein, baculovirus encoding a 6xHis-tag HBV S1/S2 protein sequence was used to infect the insect cell at a Multiplicity of Infection (MOI) = 2, when the insect cell density reached 2 ~ 2.5 *10⁶ cells /mL. After 72 hours of infection the cell pellet was harvested by centrifugation.

Xing J., Singh S., Zhao Y., Duan Y., Guo H., Hu C., Ma A., George R., Xing J.Z., Kalluri A., Macwan I., Patra P, & Chen J. (2017). Increasing vaccine production using pulsed ultrasound waves. PLoS ONE, 12(11), e0187048.

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Baculovirus Expression and Purification of TEP1 and LRIM1/APL1C

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All TEP1 and LRIM1/APL1C constructs were expressed using the baculovirus expression system using T.ni cells in ESF-921 media (Expression Systems LLC). TEP1 was expressed for ~60 h at 27°C, LRIM1/APL1C for ~48 h at 27°C. Conditioned medium was concentrated and diafiltrated with 250 mM NaCl, 20 mM Tris-HCl pH 7.8 using Centramate tangential flow filtration system (Pall Biosciences). The diafiltrated medium was loaded onto 5 ml Talon resin (Clontech), washed with 10 CV of 250 mM NaCl, 20 mM Tris-HCl pH 7.8 and eluted by a 20 ml gradient to 250 mM imidazole.
TEP1 eluate from Talon resin was immediately desalted on a HiTrap 26/10 column (GE Healthcare) equilibrated with 100 mM NaCl, 20 mM Tris-HCl pH 8.0, loaded onto a MonoQ column (GE Healthcare) and eluted with a linear gradient from 100–600 mM NaCl. Further purification was achieved by gel filtration (Superdex 200) equilibrated with 100 mM NaCl 20 mM Na-Hepes pH 7.5 and cation exchange on a MonoS column. LRIM1/APL1C eluate from Talon resin was desalted into 50 mM NaCl, 20 mM Tris-HCl pH 8.5 prior to MonoQ chromatography followed by final purification on gel filtration (Superdex 200) equilibrated with 100 mM NaCl 20 mM Na-Hepes pH 7.5.

Williams M., Contet A., Hou C.F., Levashina E.A, & Baxter R.H. (2019). Anopheles gambiae TEP1 forms a complex with the coiled-coil domain of LRIM1/APL1C following a conformational change in the thioester domain. PLoS ONE, 14(6), e0218203.

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ACKR3 Expression in Sf9 Cells

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ACKR3 was expressed in Sf9 cells as previously described (27 (link)). Briefly, cells were cultured in ESF921 media (Expression Systems) at 27°C with shaking at 140 rpm. The Bac-to-Bac Baculovirus Expression System (Invitrogen) was used to produce baculovirus containing the different ACKR3 and CXCL12 variants as previously described (27 (link)). Briefly, recombinant bacmids were incubated with 3 μL of Xtreme Gene Transfection Reagent (Roche) and 100 μL of transfection medium (Expression Systems) for 30 minutes. The mixture was added to 2.5 mL of Sf9 cells at a density of 1.3 × 10⁶ cells mL⁻¹ and the cells were incubated for 96 h with 300 rpm shaking at 27°C. Cells were pelleted using centrifugation and 400 μL of the resulting supernatant (P0 stock) was used to transfect 40 mL of Sf9 cells at a density of 2.5 × 10⁶ cells mL⁻¹. After 48 h the cells were centrifuged and the resulting supernatant was stored at 4°C until further use (P1 stock). Virus titers were determined using flow cytometry staining with a phycoerythrin (PE)conjugated GP64 antibody. To initiate protein expression P1 virus corresponding to ACKR3 alone or ACKR3 and CXCL12 (co-expression) was added to Sf9 cells at a density of ~2.5 × 10⁶ cells mL⁻¹ at a multiplicity of infection of 5. Cells were grown for 48h at 27°C with shaking at 140 rpm prior to experiments.

Gustavsson M., Dyer D.P., Zhao C, & Handel T.M. (2019). Kinetics of CXCL12 binding to atypical chemokine receptor 3 reveal a role for the receptor N-terminus in chemokine binding. Science signaling, 12(598), eaaw3657.

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Recombinant Expression of BCL6 and SIAH1

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The human wild-type and mutant versions of BCL6 (Uniprot entry: A5PL18, residue 5–129 or 5–360) and SIAH1 variants (Uniprot Entry: Q8IUQ4, residue 90–282 (substrate binding domain, SBD) or full length), were cloned in pAC-derived vectors³². Baculovirus for protein expression (Invitrogen) was generated by transfection into Spodoptera frugiperda (Sf9) cells at a density of 0.9 × 10⁶ cells/mL grown in ESF 921 media (Expression Systems), followed by three rounds of infection in Sf9 cells to increase viral titer. Recombinant proteins were expressed and purified as N-terminal His₆ C-terminal Spy (wild-type and mutant versions of BCL6⁵⁻³⁶⁰), N-terminal Strep II-Avi (BCL6⁵⁻¹²⁹, BCL6⁵⁻³⁶⁰, and SIAH1^SBD), and N-terminal Flag (SIAH1^FL) fusions in Trichoplusiani High Five insect cells using the baculovirus expression system (Invitrogen) as described previously³³.

Słabicki M., Yoon H., Koeppel J., Nitsch L., Roy Burman S.S., Di Genua C., Donovan K.A., Sperling A.S., Hunkeler M., Tsai J.M., Sharma R., Guirguis A., Zou C., Chudasama P., Gasser J.A., Miller P.G., Scholl C., Fröhling S., Nowak R.P., Fischer E.S, & Ebert B.L. (2020). Small molecule-induced polymerization triggers degradation of BCL6. Nature, 588(7836), 164-168.

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Purification and stimulation of GPCR complexes

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Commonly used reagents and cell culture consumables were purchased from Sigma-Aldrich or local vendors unless specified otherwise. E. coli expression constructs for βarr1 and 2, Fab30, ScFv30 and βarr1^245C/βarr2^246C (harboring C59V, L68C, C125S, C140L, C150V, C242V, C251V, C269S mutations) are described earlier, and these proteins were purified using previously described protocols (Kumari et al., 2016 (link); Kumari et al., 2017b ). Expression constructs for β₂V₂R, GRK2, V₂R, and their purification details have also been published previously (Kumari et al., 2016 (link); Kumari et al., 2017b ). Briefly, FLAG-β₂V₂R and FLAG-V₂R were co-expressed with GRK2 in Sf9 cells (cultured in ESF921 media from Expression Systems), and 60–66h post-infection; cells were stimulated with indicated ligands and harvested by centrifugation.

Ghosh E., Dwivedi H., Baidya M., Srivastava A., Kumari P., Stepniewski T., Kim H.R., Lee M.H., van Gastel J., Chaturvedi M., Roy D., Pandey S., Maharana J., Guixà‐Gonzàlez R., Luttrell L.M., Chung K.Y., Dutta S., Selent J, & Shukla A.K. (2019). Conformational biosensors and domain-swapping reveal structural and functional differences between the β-arrestin isoforms. Cell reports, 28(13), 3287-3299.e6.

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Suspension Culture of Sf9 Cells

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Suspension cultures of Sf9 cells (Cat# 94-001S), growing in ESF 921 media (Cat# 96-001), were purchased from Expression Systems. Cultures were passaged every two days and strictly maintained in log-phase growth for ~15 generations, at an optimal cell density of 1–5 × 10⁶ cells/ml (27 °C, 135 rpm (~1×g); Eppendorf Innova Model 2300 large capacity platform shaker). Log-phase Sf9 cells are typically round in shape, uniform in size and do not form cell clumps. Over growth was carefully avoided. Morphological changes in Sf9 cells such as swelling (accumulation of tetraploid cells) and clumping (environmentally-stressed cells) are indications that a new culture should be started.

Sinha N.K, & Bass B.L. (2017). Overexpression and purification of Dicer and accessory proteins for biochemical and structural studies. Methods (San Diego, Calif.), 126, 54-65.

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Recombinant COBRA Protein Expression

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Y2 COBRA was cloned into the pBacPAK8 vector in frame with an N-terminal gp67 signal sequence and C-terminal thrombin cleavage site, T4 fibritin domain, and hexahistidine/StrepTag II tags. The construct design results in predicted vector supplied sequences of AATNA and LVPRGSPGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGHHHHHHGGSWSHPQFEK at the N- and C-termini, respectively. Baculovirus was generated using the flashBac™ kit according to the manufacturer’s instructions (Mirus Bio). The protein was expressed in 2 L of Sf9 cells at 2×10⁶ cells/mL maintained in ESF921 media (Expression Systems) by adding 25 mL virus per liter of culture. The media was harvested after 3 days, pH adjusted with NaCl and Tris pH 8, and stored at −20°C. Prior to purification, the thawed media was filtered and concentrated to 150-200 mL by tangential flow with a Vivaflow® 200 (Sartorius). The resulting sample was diluted with an equal volume of Buffer A, filtered, and loaded onto a 5 mL HisTrap column (GE Healthcare). The column was washed with Buffer A and the protein eluted with a gradient to Buffer B. The protein was pooled, concentrated, and supplemented with 5% glycerol prior to flash freezing and storage at −80 °C.

Nagashima K., Dzimianski J.V., Han J., Abbadi N., Gingerich A.D., Royer F., O’Rourke S., Sautto G.A., Ross T.M., Ward A.B., DuBois R.M, & Mousa J.J. (2022). The pre-existing human antibody repertoire to computationally optimized influenza H1 hemagglutinin vaccines. Journal of immunology (Baltimore, Md. : 1950), 209(1), 5-15.

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Maintenance of Cell Lines for Receptor Expression

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Tni and Sf9 cells (Expression Systems) were maintained in ESF-921 media (Expression Systems) at 27°C. Flp-In Chinese hamster ovary (CHO) (Thermo Fisher Scientific) cells stably expressing human M₄ mAChR or mutant constructs were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen) containing 5% fetal bovine serum (FBS; ThermoTrace) and 0.6 μg/ml of Hygromycin (Roche) in a humidified incubator (37°C, 5% CO₂, 95% O₂). HEK293A cells were grown in DMEM supplemented with 5% FBS at 37°C in 5% CO₂. Cell lines were authenticated by vendor and confirmed negative for mycoplasma contamination using the Lonza MycoAlert Mycoplasma Detection Kit (#LT07-318).

Vuckovic Z., Wang J., Pham V., Mobbs J.I., Belousoff M.J., Bhattarai A., Burger W.A., Thompson G., Yeasmin M., Nawaratne V., Leach K., van der Westhuizen E.T., Khajehali E., Liang Y.L., Glukhova A., Wootten D., Lindsley C.W., Tobin A., Sexton P., Danev R., Valant C., Miao Y., Christopoulos A, & Thal D.M. (2023). Pharmacological hallmarks of allostery at the M4 muscarinic receptor elucidated through structure and dynamics. eLife, 12, e83477.

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