Purified His-tagged MYC (TP760019) was purchased from Origene Inc. GST-tagged TRIB3 (10731-H09B) and KDC domains, GST-tagged MAX (12885-H20B) and His-tagged MAX (12269-H08H) were purchased from Sinobiological Inc. The recombinant proteins UBE3B (His-tagged), UBE3B-C (His-tagged), UBE3B R346Q, and UBE3B C1036A were produced in FreestyleTM 293-F cells (Life Technology, Carlsbad, CA, USA) following transient transfection with 1 mg/mL DNA at a DNA/PEI ratio of 1:2.5 (PEI, Polyscience). Proteins were purified from culture supernatants using Ni-NTA-Sepharose (GE Healthcare) and dialyzed against PBS. Purified proteins were loaded onto a Hiload 16/600 Superdex 200 gel filtration column (GE Healthcare) to remove aggregates. Purified proteins were concentrated to 4 mg/mL by a 10-kDa Centrifugal Filter Unit (Millipore, Burlington, MA, USA) and filtered by a 0.22-μm Millipore filter.
Hiload 16 600 superdex 200 gel filtration column
Manufactured by GE Healthcare
The Hiload 16/600 Superdex 200 gel filtration column is a laboratory equipment used for size-exclusion chromatography. It is designed to separate and purify biomolecules based on their size and molecular weight. The column has a bed volume of 120 mL and is compatible with a wide range of aqueous buffers and organic solvents, making it suitable for a variety of applications in the field of biochemistry and protein purification.
Automatically generated - may contain errors
Lab products found in correlation
Freestyle 293 f cells, by Thermo Fisher Scientific (2 mentions)
Polyethylenimine (pei), by Polysciences (2 mentions)
Protein g sepharose, by GE Healthcare (2 mentions)
Ni nta sepharose, by GE Healthcare (1 mentions)
10 kda centrifugal filter unit, by Merck Group (1 mentions)
Nanodrop 2000 2000c spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Whatman filter paper, by Cytiva (1 mentions)
Amicon ultra centrifugal filter unit, by Merck Group (1 mentions)
Model 705 sonic dismembrator, by Thermo Fisher Scientific (1 mentions)
Centrifugal filter unit, by Merck Group (1 mentions)
Ni2 nta agarose, by Qiagen (1 mentions)
Expi293f cells, by Thermo Fisher Scientific (1 mentions)
Yarra 3 μm sec 3000 column, by Phenomenex (1 mentions)
Protein a sepharose, by Thermo Fisher Scientific (1 mentions)
5 protocols using hiload 16 600 superdex 200 gel filtration column
1
Purification of Recombinant Proteins
2
Purification and Characterization of Pea Vicilin
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Pisum sativum seeds (10 g) were soaked and homogenized in 100 mL of phosphate buffer (100 mM, pH 7.0) and centrifuged at 10 000 ×g for 15 minutes. Supernatant was collected and filtered through Whatman filter paper (11 µm). The clear solution was subjected to 60% ammonium sulfate saturation under cold conditions and centrifuged at 3000 ×g for 5 minutes. Supernatant was collected and salts were removed by using a dialysis membrane of 3.5 kDa MWCO (Spectra/Por 3; Catalog no. 132724) in the same extraction buffer. Dialyzed sample was loaded onto anion exchanger-Hi Trap Q FF column with 100 mM phosphate buffer (pH 7.0) at a flow rate of 0.2 mL/min and eluted with NaCl gradient of 0-1 M in the same buffer. Purified vicilin was subjected to Hi-load 16/600 Superdex 200 gel filtration column (GE Healthcare) using the same buffer and a UV detector (280 nm) was used for the recording of eluent absorbance. Fractions with maximum protein quantities were pooled together and were loaded on SDS-PAGE under both reduced and non-reduced conditions to analyze protein banding patterns along the protein ladder (Thermo ScientificTM catalog number 26616).22
Protein quantification was done by nanodrop (NanoDrop™ 2000/2000c Spectrophotometers).
Protein quantification was done by nanodrop (NanoDrop™ 2000/2000c Spectrophotometers).
3
DhaA115 Enzyme Purification from E. coli
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The DhaA115 enzyme was overproduced in Escherichia coli, as previously described. Briefly, DhaA115 was overexpressed in E. coli BL21(DE3) with induction by 0.5 mM IPTG at 20 °C for 16 hours. The cells were harvested by centrifugation at 11 806g at 4 °C for 10 min. The pellet was re-suspended in purification buffer A (500 mM NaCl, 10 mM imidazole, 20 mM potassium phosphate buffer pH 7.5) and sonicated using a Sonic Dismembrator Model 705 (Fisher Scientific, USA) in 3 cycles, each of 2 min (5 s pulse/5 s pause) with amplitude 50%. Disrupted cells were centrifuged at 21 000g at 4 °C for 1 h. His-tagged DhaA115 protein was purified on a Ni-chelating column (Ni-NTA Superflow cartridge) equilibrated in purification buffer A. The affinity-purified enzyme was eluted by a purification buffer A supplemented with 300 mM imidazole. The eluted protein was further purified by size-exclusion chromatography on a HiLoad 16/600 Superdex 200 gel filtration column (GE Healthcare) equilibrated in GF buffer (50 mM NaCl, 10 mM Tris pH 8.0). Peak fractions were pooled and concentrated with an Amicon Ultra centrifugal filter unit (Merck Millipore Ltd) to a final concentration of 11.5 mg ml−1. Protein concentration was measured on a DeNovixR® DS-11 Spectrophotometer (DeNovix Inc., USA).
4
Recombinant Antibody Generation Protocol
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IgGs were generated from scFv genes as described25 (link). Briefly, VH genes were amplified using PCR and cloned into EcoRI- and NheI- digested pFUSEss-CHIg-hG1 vector. Similarly, VL genes were amplified, digested with EcoRI as well as BsiwI and cloned into pFUSE2ss-CLIg-hk. For V gene cloning, pFUSE2ss-CLIg-hL2 vector and PCR products were digested with EcoRI and AvrII.
Recombinant antibodies were produced in FreestyleTM 293-F cells (Life Technology, Carlsbad, CA, USA) following transient transfection with 1 mg/mL DNA at a DNA/PEI ratio of 1:2.5 (PEI, Polyscience) as previously described25 (link). Antibodies were purified from culture supernatants using protein G-Sepharose (GE Healthcare) and dialyzed against PBS. Purified antibodies were loaded onto a Hiload 16/600 Superdex 200 gel filtration column (GE Healthcare) to remove aggregates, followed by analyses of the ‘monomeric’ antibodies using SuperdexTM 200 Increase 3.2/300 (GE Healthcare). Purified proteins were concentrated to 4 mg/mL by a 10 kDa Centrifugal Filter Unit (Millipore, Burlington, MA, USA), filtered by 0.22 µm Millipore filter.
Recombinant antibodies were produced in FreestyleTM 293-F cells (Life Technology, Carlsbad, CA, USA) following transient transfection with 1 mg/mL DNA at a DNA/PEI ratio of 1:2.5 (PEI, Polyscience) as previously described25 (link). Antibodies were purified from culture supernatants using protein G-Sepharose (GE Healthcare) and dialyzed against PBS. Purified antibodies were loaded onto a Hiload 16/600 Superdex 200 gel filtration column (GE Healthcare) to remove aggregates, followed by analyses of the ‘monomeric’ antibodies using SuperdexTM 200 Increase 3.2/300 (GE Healthcare). Purified proteins were concentrated to 4 mg/mL by a 10 kDa Centrifugal Filter Unit (Millipore, Burlington, MA, USA), filtered by 0.22 µm Millipore filter.
5
Recombinant Mouse MOG Protein Purification
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Recombinant mouse MOG (extracellular domain) was purified from the culture supernatant of baculovirally infected High -Five cells using Ni2+-NTA agarose (QIAGEN, Germantown, MD, USA) as described previously.56 (link) All Fc fusion proteins and the ch8-18C5 antibody were expressed in Expi293F cells (Thermo Fisher Scientific) following transient transfection with the GIBCO Expi293 expression system kit (Thermo Fisher Scientific) and were purified from culture supernatants using protein G-Sepharose (GE Healthcare) or, for MOG-Seldeg-FcRn, protein A-Sepharose (Invitrogen). Recombinant proteins were eluted from the columns using 50 mM diethylamine/150 mM NaCl and immediately neutralized using 2 M Tris-HCl pH 7.0 followed by dialysis against phosphate-buffered saline (PBS). Recombinant Fc fusion proteins were further purified using a Hiload 16/600 Superdex 200 gel filtration column (GE Healthcare). Purified proteins were analyzed by SDS-PAGE and by size exclusion chromatography (SEC) using a Phenomenex Yarra 3 μm SEC-3000 column (Phenomenex, 00H-4513-K0, Torrance, CA, USA).
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