Biosep sec s3000 column

Manufactured by Phenomenex

Sourced in United States, Germany

The BioSep SEC-s3000 column is a size exclusion chromatography (SEC) column used for the separation and analysis of biomolecules such as proteins, peptides, and other macromolecules. The column is designed to provide efficient and reliable separation of these molecules based on their molecular size and shape.

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Lab products found in correlation

Pe 343 polarimeter, by PerkinElmer (4 mentions) 20 μl loop, by Waters Corporation (4 mentions) 20 l loop, by Waters Corporation (3 mentions) Amicon 10 kda mwco filtration unit, by Merck Group (2 mentions) Ni nta bead, by Qiagen (2 mentions) Isopropyl β d 1 thiogalactopyranoside (iptg), by Thermo Fisher Scientific (2 mentions) Cary 100 uv visible uv vis spectrophotometer, by Agilent Technologies (2 mentions) Minidawn treos line, by Wyatt Technology (2 mentions) Astra 6, by Wyatt Technology (2 mentions) 1260 infinity hplc system, by Agilent Technologies (2 mentions) Double sector jms ax505ha mass spectrometer, by JEOL (1 mentions) Arsenazo 3, by Merck Group (1 mentions) 300 instrument, by Bruker (1 mentions) Diferric holo transferrin, by Merck Group (1 mentions) Bovine serum albumin (bsa), by Merck Group (1 mentions) Heleos 2, by Wyatt Technology (1 mentions) Optilab t rex, by Wyatt Technology (1 mentions) Akta purifier, by GE Healthcare (1 mentions) Minidawn malls detector, by Wyatt Technology (1 mentions) Optilab t rex refractive index detector, by Wyatt Technology (1 mentions)

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BioSep s3000 (2 citations)

29 protocols using biosep sec s3000 column

Production of αCD8, αHLA-I, and αCD47 Nanobodies

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Monomeric and dimeric variants of two human αCD8 nanobody clones (αCD8^M, αCD8^D-1, αCD8^D-2) and dimeric variants of αHLA class I (specific for the β2M subunit of HLA class I, αHLA-I^D) and mouse αCD47 (αCD47^D) nanobody clones were generated as follows: Escherichia coli WK6 cells were transformed with the pHEN6 expression vector (for production of the αCD8^M nanobody) and Escherichia coli BL21 cells were transformed with the pET22b expression vector (for production of the αCD8^D, αHLA-I^D and αCD47^D nanobodies) encoding the relevant nanobody sequence, followed by an LPETGG-6xH sequence. αCD8^D nanobodies are formed by coupling two monomeric αCD8 domains with a flexible GC-rich linker. Protein production was induced with IPTG (Thermo Fisher Scientific) and recombinant proteins were isolated from the periplasmic fraction using Ni-NTA beads (Qiagen). Following washing and subsequent elution with 50 mM Tris (pH 8), 150 mM NaCl, 500 mM imidazole, samples were purified by gel filtration chromatography on a Phenomenex Biosep SEC-S3000 column in phosphate-buffered saline (PBS) and material was concentrated using an Amicon 10 kDa MWCO filtration unit (Millipore). Nanobodies were stored at −80 °C until further use.

van der Leun A.M., Hoekstra M.E., Reinalda L., Scheele C.L., Toebes M., van de Graaff M.J., Chen L.Y., Li H., Bercovich A., Lubling Y., David E., Thommen D.S., Tanay A., van Rheenen J., Amit I., van Kasteren S.I, & Schumacher T.N. (2021). Single Cell Analysis of Regions of Interest (SCARI) using a photo-sensitive tag. Nature chemical biology, 17(11), 1139-1147.

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Characterization of Metal-Protein Complexes

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¹H and ¹³C NMR spectra were obtained using a Bruker 300 instrument, and chemical shifts are reported in ppm on the δ scale relative to TMS or solvent. Fast atom bombardment (FAB) or Electrospray iodization (ESI) high resolution mass spectra (HRMS) were obtained on JEOL double sector JMS-AX505HA mass spectrometer (University of Notre Dame, IN). Size-exclusion HPLC (SE-HPLC) chromatograms were obtained on Agilent 1200 equipped with a diode array detector and an in-line IN/US γ-Ram Model 2 radiodetector (Tampa, FL), fitted with BioSep-SEC S 3000 column (Phenomenex, Torrance, CA). All absorbance measurements for the protein concentration and ligand protein ratio were obtained on an Agilent 8453 diode array spectrophotometer equipped with an 8-cell transport system (designed for 1-cm cells). Arsenazo III (AAIII, 2,2-(1,8-dihydroxy-3,6-disulfonaphthylene-2,7-bisazo) bis-benzenearsonic acid), copper atomic absorption standard solution, and diferric (holo) transferrin were purchased from Sigma-Aldrich (St. Louis, MO) and used as received. C-DOTA analogues were purchased from Macrocyclics (Dallas, TX).

Kang C.S., Wu N., Chen Y., Sun X., Bandara N., Liu D., Lewis M., Rogers B, & Chong H.S. (2015). Transferrin Conjugates of Triazacyclononane-Based Bifunctional NE3TA Chelates for PET Imaging: Synthesis, Cu-64 Radiolabeling, and In vitro and In vivo evaluation. Journal of inorganic biochemistry, 154, 60-66.

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Molecular Characterization of Proteinoids

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Gel permeation chromatography (GPC) was used in order to analyze the molecular weight and polydispersity index of the proteinoids, as described earlier⁸. GPC spectra were obtained with a Waters Spectra Series P100 isocratic high performance liquid chromatography (HPLC) pump with UV/vis detector, ERMA ERC-7510 refractive index detector, and a Rheodyne (Coatati, CA) injection valve with a 20 µL loop (Waters, MA). Samples were eluted with super-pure HPLC water through a linear BioSep SEC-s-3000 column (Phenomenex) at 1 mL/min flow rate. Poly(ethylene glycol) standards (Polymer Standards Service, Silver Spring, MD, USA) with molecular weights between 100–450,000 Da, HSA (67 kDa), and bovine plasma fibrinogen (340 kDa) were used to determine the molecular weights using Clarity chromatography software. A PE 343 polarimeter (PerkinElmer, MA, USA) was used to determine the optical activity of the proteinoids. All measurements were performed at 25 °C in water at 589 nm.

Sasson E., Pinhasi R.V., Margel S, & Klipcan L. (2020). Engineering and use of proteinoid polymers and nanocapsules containing agrochemicals. Scientific Reports, 10, 9171.

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Molecular Mass Determination of LapD Protein

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Molecular masses of LapD protein samples were determined using size exclusion chromatography coupled with static multiangle light scattering (SEC-MALS) (46 (link)). Purified protein at concentrations between 1 and 15 mg/ml (20 to 300 μM) was subjected to gel filtration on a Bio Sep-SEC-s 3000 column (Phenomenex, Torrance, CA) that was equilibrated in MALS buffer (25 mM Tris-HCl [pH 7.5], 150 mM NaCl). For samples with c-di-GMP in the mobile phase, 50 μM c-di-GMP was added to the MALS buffer. The SEC was coupled to a static 18-angle light-scattering detector (DAWN HELEOS-II) and a refractive index detector (Optilab T-rEX; Wyatt Technology, Goleta, CA). Data were collected at 25°C each second for 30 min at a flow rate of 1 ml/min. Data analysis was carried out using the program ASTRA V. The detectors were normalized using a sample of 5 mg/ml bovine serum albumin (BSA) (monomeric fraction; Sigma-Aldrich, St. Louis, MO).

Kitts G., Giglio K.M., Zamorano-Sánchez D., Park J.H., Townsley L., Cooley R.B., Wucher B.R., Klose K.E., Nadell C.D., Yildiz F.H, & Sondermann H. (2019). A Conserved Regulatory Circuit Controls Large Adhesins in Vibrio cholerae. mBio, 10(6), e02822-19.

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Molecular Mass Analysis of NS5A

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MALLS was carried out both in batch mode using a syringe pump set to 0.1 ml/min and through size-exclusion chromatography (SEC) using a BioSep-SEC S3000 column (Phenomenex) equilibrated in a buffer containing 25 mM Tris-HCl, pH 8.0, 250 mM NaCl, 10% glycerol, 0.05% Tween-20, 5% DMSO and in the presence and absence of 5 mM β-mercaptoethanol. The column was run at a flow rate of 0.35 ml/min on an AKTApurifier (GE Healthcare) coupled to a miniDawn MALLS detector and an Optilab T-rEX refractive index detector (Wyatt Technology). The molecular masses of bovine serum albumin (used as a control) and NS5A^33–202 were calculated using a dn/dc value of 0.185. NS5A^33–202 was run at a range of concentrations from 0.3–4 mg/ml. The overall weight-average molar mass for each sample was determined from the concentrations of each molar mass at all points by use of ASTRA 5 software (Wyatt Technologies).

Ascher D.B., Wielens J., Nero T.L., Doughty L., Morton C.J, & Parker M.W. (2014). Potent hepatitis C inhibitors bind directly to NS5A and reduce its affinity for RNA. Scientific Reports, 4, 4765.

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Antibody-SOD Ferritin Nanocarrier Conjugation

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Heterobifunctional crosslinkers SATA and SMCC were used for conjugation of antibody and SOD to horse spleen Ft as described earlier [33 ]. SATA was added at 1:5 M ratio of antibody or SOD to SATA for 30 min at rt. SATA was then de-protected using 10% 0.5 M hydroxylamine for 2 h at room temperature. Ft was added at 1:150 M ratio of Ft (based on Ft 24-mer molar concentration) to SMCC for introducing maleimide groups. Antibody and SOD conjugation to Ft was carried out at 1:10 M ratio of Ft to mixture of antibody and SOD for 1 h at room temperature. Unbound reagents were removed by desalting columns. The nanocarriers were purified by size-exclusion high-performance liquid chromatography (SEC-HPLC) using BioSep SEC-s3000 column (Phenomenex, Torrance, CA). The samples were purified by isocratic method with PBS pH 7.4 to remove free antibody [33 ]. For experiments with drug-free ferritin nanocages, antibodies were conjugated to ferritin in the absence of SOD.

Shuvaev V.V., Khoshnejad M., Pulsipher K.W., Kiseleva R.Y., Arguiri E., Cheung-Lau J.C., LeFort K.M., Christofidou-Solomidou M., Stan R.V., Dmochowski I.J, & Muzykantov V.R. (2018). Spatially controlled assembly of affinity ligand and enzyme cargo enables targeting ferritin nanocarriers to caveolae. Biomaterials, 185, 348-359.

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Peptide-MHC I Complex Exchange Assay

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To initiate peptide exchange, 0.5 µM peptide–MHC I complex was incubated with 50 µM exchange peptide in 110 µl PBS under defined exchange conditions. After incubation exchange solutions were centrifuged at 14,000 g for 1 min at room temperature, and subsequently, the supernatant was analyzed by gel filtration on a Shimadzu Prominence HPLC system equipped with a 300 × 7.8 mm BioSep SEC–s3000 column (Phenomenex) using PBS as mobile phase. Data were processed and analyzed using Shimadzu LabSolutions software (version 5.85).

Luimstra J.J., Garstka M.A., Roex M.C., Redeker A., Janssen G.M., van Veelen P.A., Arens R., Falkenburg J.H., Neefjes J, & Ovaa H. (2018). A flexible MHC class I multimer loading system for large-scale detection of antigen-specific T cells. The Journal of Experimental Medicine, 215(5), 1493-1504.

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Production of αCD8, αHLA-I, and αCD47 Nanobodies

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Purification of Membrane-bound CybB Proteins

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Membranes were thawed and solubilized in 1% OGNG (Octyl Glucose Neopentyl Glycol) (Anatrace) for 2 hours. Insoluble material was cleared by ultracentrifugation for 30 min. at 40.000 rpm in a Beckman Ti70.1 rotor. The supernatant was incubated at 4°C with Ni²⁺-charged Profinity (Bio-Rad) IMAC resin for 2 h with gentle rotation (5 mM Imidazole was added after 1 h of incubation). The sample was loaded on a Econo-Pac disposable gravity flow column (Bio-Rad). The settled resin was washed with 15 CV IMAC wash buffer 1 (10 mM HEPES pH 7.0, 200 mM NaCl, 50 mM Imidazole, 5% glycerol, 0.1% OGNG) and 15 CV wash buffer 2 (10 mM HEPES pH 7.0, 200 mM NaCl, 5% glycerol, 0.1% OGNG). CybB/CybB_SeMet was eluted with 3x 0.75 CV elution buffer (10 mM HEPES pH 7.0, 200 mM NaCl, 100 mM EDTA, 5% glycerol, 0.1% OGNG), concentrated to 0.5 ml using Vivaspin concentration devices with 50 kD cutoff (Sartorius) and subjected to size exclusion chromatography (10 mM HEPES pH 7.0, 200 mM NaCl, 5% glycerol, 0.1% OGNG) on a 30 ml BIOSEP-SEC-S3000 column (Phenomenex) connected to a Shimadzu Prominence HPLC system. Fractions representing the CybB/CybB_SeMet peak were pooled, concentrated to 10-12 mg/ml (CybB) and 11 mg/ml (CybB_SeMet) with 50 kD concentrator cut-off, and flash-frozen in liquid nitrogen.

Lundgren C.A., Sjöstrand D., Biner O., Bennett M., Rudling A., Johansson A.L., Brzezinski P., Carlsson J., von Ballmoos C, & Högbom M. (2018). Scavenging of superoxide by a membrane bound superoxide oxidase. Nature chemical biology, 14(8), 788-793.

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Characterization of Proteinoid Polymers

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The molecular weights and polydispersity index of the proteinoid polymer were determined by gel permeation chromatography (GPC) consisting of a Waters Spectra Series P100 isocratic HPLC pump with ERMA ERC-7510 refractive index detector and Rheodyne injection valve (Coatati, CA, USA) with a 20 µL loop (Waters, Milford, MA, USA). The sample was dissolved with super-pure HPLC water (Sigma) through linear BioSep SEC-s3000 column (Phenomenex, Torrence, CA, USA) at a flow rate of 1 mL/min. The molecular weight of the proteinoid was determined relative to poly(ethylene glycol) (PEG) standards (Polymer Standards Service-USA, Silver Spring, MD, USA) with a molecular weight range of 0.10−450 kDa, bovine plasma fibrinogen (340 kDa), and human serum albumin (67 kDa) using Clarity chromatography software (DataApex, Prague, Czech Republic).
The absorption spectrum of the proteinoid was obtained by using a Cary 100 UV-Visible (UV-Vis) spectrophotometer (Agilent Technologies, Santa Clara, CA, USA). Fourier transform infrared (FTIR) measurement of the proteinoid was performed by the Attenuated Total Reflectance (ATR) technique using a Bruker Alpha FTIR QuickSnapTM sampling module equipped with a Platinum ATR diamond module (Bruker Optik GmbH, Ettlingen, Germany).

Hadad E., Rudnick-Glick S., Itzhaki E., Avivi M.Y., Grinberg I., Elias Y, & Margel S. (2020). Engineering of Doxorubicin-Encapsulating and TRAIL-Conjugated Poly(RGD) Proteinoid Nanocapsules for Drug Delivery Applications. Polymers, 12(12), 2996.

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