Superdex 75 26 600

Manufactured by GE Healthcare

Sourced in Sweden

Superdex 75 26/600 is a size exclusion chromatography column used for the separation and purification of proteins, peptides, and other biomolecules based on their molecular size. The column has a bed volume of 320 ml and is designed for preparative-scale separations.

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Lab products found in correlation

Superdex 200 26 600, by GE Healthcare (2 mentions) Thermomixer, by Eppendorf (1 mentions) Fluostar omega, by BMG Labtech (1 mentions) Costar 96 well plate, by Corning (1 mentions) Freestyle 293 f, by Thermo Fisher Scientific (1 mentions) Ni nta imac sepharose 6 fast flow resin, by GE Healthcare (1 mentions) Polyethyleneimine, by Polysciences (1 mentions) Pet28a, by GenScript (1 mentions) Superdex 200 26 600 column, by GE Healthcare (1 mentions) Vivaspin protein concentrator, by GE Healthcare (1 mentions) Thrombin, by Merck Group (1 mentions)

4 protocols using superdex 75 26 600

Fibrillation of Variant β2-Microglobulin Proteins

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To generate fibrils of WT- β₂m, β₂m-ΔN6, β₂m-D76N and β₂m-V27M for ThT and EM studies, an aliquot of each frozen protein was thawed and passed through a Superdex 75 26/600 (GE healthcare) size exclusion chromatography column, previously equilibrated with fibrillation buffer (25 mM sodium phosphate, 115 mM NaCl, pH 6.2). Each eluted monomeric peak was collected and adjusted to the final β₂m monomer concentration desired and used immediately. For ThT fibril growth assay, five replicate reactions were set for each protein in Costar 96-well plates (Corning) using a Fluostar Omega (BMG Labtech) plate-reader. Reaction volumes were 100 µL, with 40 µM of each protein and 10 µM ThT in the fibrillation buffer. The ThT reactions were run for 100 h at 37 °C with 600 rpm shaking for 5 min at 6 min intervals and the fluorescence for each individual run was normalised to an arbitrary scale of 0 to 1 for plotting. For structure determination, 20 µM (β₂m-ΔN6 and β₂m-V27M) or 40 µM (β₂m-D76N) of protein in 1 mL fibrillation reactions in 1.5 mL Eppendorf tubes were incubated at 37 °C with continuous shaking at 600 rpm in a benchtop Eppendorf thermomixer. Fibrils were visible by negative stain EM after 72 h.

Wilkinson M., Gallardo R.U., Martinez R.M., Guthertz N., So M., Aubrey L.D., Radford S.E, & Ranson N.A. (2023). Disease-relevant β2-microglobulin variants share a common amyloid fold. Nature Communications, 14, 1190.

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Recombinant Antibody Fab Production

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Recombinant antibody Fabs were transiently expressed in FreeStyleTM 293F (Invitrogen) suspension cultures by co-transfection of pVRC8400 plasmids containing expression constructs for light chain and Fab heavy chain using polyethyleneimine (Polysciences). Cell growth was harvested on day 6 post transfection. eOD-GT6 was also produced in FreeStyleTM 293F (Invitrogen) suspension cultures by transient transfection using polyethyleneimine (Polysciences) of a pHLSec plasmid containing mammalian codon-optimized eOD-GT6 with a C-terminal Avi and His6x affinity tag. Proteins were harvested from the supernatant after 96 h.
The secreted proteins were purified by using Ni-NTA IMAC Sepharose 6 Fast Flow resin (GE Healthcare) nickel affinity chromatography followed by size exclusion chromatography (SEC) using a Superdex 200 26/600 (Fabs) or Superdex 75 26/600 (eOD) column (GE Healthcare) in 10 mM Tris pH 8.0, 150 mM NaCl SEC buffer. Peak fractions containing Fabs or eOD-GT6 were pooled. Protein purity was analyzed by SDS-PAGE and concentrated where possible to ~10 mg/mL. BG505-SOSIP was requested from the Vaccine Research Center at the National Institute of Health (47 (link)).

Sheng Z., Bimela J.S., Katsamba P.S., Patel S.D., Guo Y., Zhao H., Guo Y., Kwong P.D, & Shapiro L. (2022). Structural Basis of Antibody Conformation and Stability Modulation by Framework Somatic Hypermutation. Frontiers in Immunology, 12, 811632.

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Recombinant PD-L1 and PD-1 Constructs

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Constructs encoding the extracellular regions of human PD-L1 D1D2 (18–237), PD-L D1 (18–134), and PD-1 (33–150) with a C-terminal avitag (GGLNDIFEAQKIEWHE) were synthesized and cloned into pET28a by GenScript. A C-terminal avitag construct of PD-L1 D1D2 (18–237) was produced by mutagenesis (https://openwetware.org/wiki/%27Round-the-horn_site-directed_mutagenesis, accessed February 2, 2022) (58 (link)). PD-L1 D2 (130–239) was cloned into the pLEICS-05 vector by PROTEX (University of Leicester). PD-L1 and PD-1 constructs were expressed and refolded as previously reported (6 (link)). Proteins were purified using a Superdex 75 26/600 gel filtration column (GE Healthcare) equilibrated in 25 mM KH₂PO₄, pH 7.4, 25 mM NaCl, and 10 μM EDTA. Isotopically labeled PD-L1 D1 was expressed in modified Spizizen’s minimal media (59 (link), 60 (link)), incorporating ¹⁵N-NH₄Cl and ¹³C-glucose, and was refolded and purified as aforementioned.

Kang-Pettinger T., Walker K., Brown R., Cowan R., Wright H., Baravalle R., Waters L.C., Muskett F.W., Bowler M.W., Sawmynaden K., Coombs P.J., Carr M.D, & Hall G. (2022). Identification, binding, and structural characterization of single domain anti-PD-L1 antibodies inhibitory of immune regulatory proteins PD-1 and CD80. The Journal of Biological Chemistry, 299(1), 102769.

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Purification of Bri2 BRICHOS Oligomers

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IMAC (immobilized metal affinity chromatography) purified NT*-Bri2 BRICHOS was concentrated to 3.4 mg/mL with a Vivaspin protein concentrator (10 kDa cut-off, GE Healthcare) at 4000×g and 4 °C. Then, the different rh NT*-Bri2 BRICHOS oligomeric species were isolated by size exclusion chromatography (SEC) using a Superdex 200 26/600 column (GE Healthcare, Uppsala, Sweden), which was equilibrated with Buffer B (20 mM NaP_i buffer pH 8.0 containing 0.2 mM EDTA), and an ÄKTA Explorer liquid chromatographic system (GE Healthcare, Uppsala, Sweden). The individual peaks corresponding to the differently sized oligomeric species were narrowly collected. Then, the rh NT*-Bri2 BRICHOS species were cleaved separately with 1: 600 thrombin (w/w, Merck) in a cold room overnight. After cleavage, the NT* tag was removed by reapplying the sample onto an IMAC column with buffer B (reverse IMAC), where rh Bri2 BRICHOS was found in the flow-through, and the NT*-tag bound to the column was eluted with buffer B containing 200 mM imidazol. To polish the Bri2 BRICHOS species, they were further purified with SEC one more time, using a Superdex 200 26/600 (for oligomer, tetramer, and dimer), or a Superdex 75 26/600 (for monomer) column (GE Healthcare, Uppsala, Sweden). The purified Bri2-BRICHOS species were stored at −20 °C.

Schmuck B., Chen G., Pelcman J., Kronqvist N., Rising A, & Johansson J. (2021). Expression of the human molecular chaperone domain Bri2 BRICHOS on a gram per liter scale with an E. coli fed-batch culture. Microbial Cell Factories, 20, 150.

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