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Gel filtration columns

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Gel filtration columns are laboratory equipment used for the separation and purification of molecules, such as proteins, nucleic acids, and other biomolecules, based on their size and shape. These columns contain a porous resin that allows smaller molecules to enter the pores, while larger molecules are excluded, enabling their separation.

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Lab products found in correlation

Sodium butyrate solution, by Merck Group (2 mentions) Freestyle 293 expression medium, by Thermo Fisher Scientific (2 mentions) 293fectin transfection reagent, by Thermo Fisher Scientific (2 mentions) Hek293f suspension cells, by Thermo Fisher Scientific (2 mentions) Histrap hp, by GE Healthcare (2 mentions) Cp100wx, by Hitachi (1 mentions) C18 reverse phase column, by Waters Corporation (1 mentions) Mini protean precast gel, by Bio-Rad (1 mentions) Histrap, by GE Healthcare (1 mentions) Trypsin, by Thermo Fisher Scientific (1 mentions) Emulsiflex c5 homogenizer, by Avestin (1 mentions) Tetramethylethylenediamine temed, by Thermo Fisher Scientific (1 mentions) Dithiothreitol (dtt), by GoldBio (1 mentions) Sodium acetate, by Thermo Fisher Scientific (1 mentions) Ammonium persulfate, by Thermo Fisher Scientific (1 mentions) Riboflavin, by Thermo Fisher Scientific (1 mentions) β mercaptoethanol, by Thermo Fisher Scientific (1 mentions)

Spelling variants of this product

S-200 gel-filtration columns (3 citations) PD-10 prepoured gel-filtration columns (2 citations)

7 protocols using gel filtration columns

1

Recombinant Protein Expression and Purification

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Example 2

Protein Expression and Purification.

The recombinant proteins expressed in Escherichia coli (E. coli) BL21 (DE3) RIPL cells were purified according to previously described protocols10, 12. Influenza HA4900 was purified from HEK293F suspension cells (Thermo Fisher Scientific®, MA) according to previous reports31. Briefly, the pAAV-ITRs-HA4900 plasmid was transiently transduced into HEK293F cells maintained in FreeStyle® 293 expression medium (Thermo Fisher Scientific®, MA) using the 293Fectin™ transfection reagent (Thermo Fisher Scientific®, MA). The cells were then incubated at 37° C. and 8% CO2 while shaking at 130 rpm overnight. After 12 h, an equal volume of fresh medium supplemented with sodium butyrate solution (enhancing protein expression, 2 nM final concentration) (Sigma-Aldrich®, MO) was added to the cells. On day 7, the pure supernatants were harvested by centrifugation and filtered over 0.22-μm filters (Sartorius Stedim Biotech, Germany). Proteins were purified using HisTrapHP and gel filtration columns (GE Healthcare™, IL).

US11155835B2. Prokaryotic-eukaryotic hybrid viral vector for delivery of large cargos of genes and proteins into human cells (2021-10-26). The Catholic University of America [US]. Inventors: Venigalla B. Rao [US], Jingen Zhu [US].
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2

Fluorescent Labeling of PHEV for Cellular Imaging

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PHEV was purified by sucrose density gradient centrifugation. The virus was first purified over 30%, 45%, and 60% sucrose cushions in an ultracentrifuge (CP100WX; Hitachi) at 80,000 × g for 4 h at 4°C, and then the virus was exchanged into PBS through cycles of concentration by centrifugation (20,000 × g, 2 h, 4°C) and dilution with PBS. Purified PHEV was freshly labeled with the lipophilic fluorescent dye DiD (4-chlorobenzenesulfonate salt; Life Technologies), a dialkylcarbocyanine analog that has markedly red-shifted fluorescence excitation and emission spectra and is widely used as a labeled tracer in living or fixed cells. Briefly, the PHEV stock solution was mixed with DiD for 10 min, and the free dye was removed using gel filtration columns (GE Healthcare) and HS buffer (2.5 mM HEPES, 145 mM NaCl). The Neuro-2a cells were infected with DiD-PHEV, and images were obtained and analyzed using a confocal microscope.
Li Z., Zhao K., Lan Y., Lv X., Hu S., Guan J., Lu H., Zhang J., Shi J., Yang Y., Song D., Gao F, & He W. (2017). Porcine Hemagglutinating Encephalomyelitis Virus Enters Neuro-2a Cells via Clathrin-Mediated Endocytosis in a Rab5-, Cholesterol-, and pH-Dependent Manner. Journal of Virology, 91(23), e01083-17.
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3

Influenza HA4900 Protein Expression and Purification

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The recombinant proteins expressed in E. coli BL21 (DE3) RIPL cells were purified according to previously described protocols (10 (link), 12 (link)). Influenza HA4900 was purified from HEK293F suspension cells (Thermo Fisher Scientific, MA) according to previous reports (31 (link)). Briefly, the pAAV-ITRs-HA4900 plasmid was transiently transduced into HEK293F cells maintained in FreeStyle 293 expression medium (Thermo Fisher Scientific, MA) using the 293fectin Transfection Reagent (Thermo Fisher Scientific, MA). The cells were then incubated at 37°C and 8% CO2 while shaking at 130 rpm overnight. After 12 hours, an equal volume of fresh medium supplemented with sodium butyrate solution (enhancing protein expression; 2 nM final concentration) (Sigma-Aldrich, MO) was added to the cells. On day 7, the culture supernatants were harvested by centrifugation and filtered over 0.22-μm filters (Sartorius Stedim Biotech, Germany). Proteins were purified using HisTrap HP and gel filtration columns (GE Healthcare, IL).
Zhu J., Tao P., Mahalingam M., Sha J., Kilgore P., Chopra A.K, & Rao V. (2019). A prokaryotic-eukaryotic hybrid viral vector for delivery of large cargos of genes and proteins into human cells. Science Advances, 5(8), eaax0064.
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4

Labeling WSSV Virions with DiD Dye

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WSSV virions were freshly labeled with the lipophilic fluorescent dye DiD (4-chlorobenzenesulfonate salt; Life Technologies) as previously described54 (link) with slight modifications. Briefly, the WSSV stock solution was mixed with DiD for 10 min and the free dye was removed using gel filtration columns (GE Healthcare) in HS buffer (2.5 mM HEPES, 145 mM NaCl). The Hpt cells were infected with DiD-WSSV (MOI of 20), and images were obtained and analyzed using an iCys laser-scanning cytometer (Beckman), which could automatically identify the target fluorescent signal events through proper contouring as described in the references55 (link)56 (link).
Chen R.Y., Shen K.L., Chen Z., Fan W.W., Xie X.L., Meng C., Chang X.J., Zheng L.B., Jeswin J., Li C.H., Wang K.J, & Liu H.P. (2016). White spot syndrome virus entry is dependent on multiple endocytic routes and strongly facilitated by Cq-GABARAP in a CME-dependent manner. Scientific Reports, 6, 28694.
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5

Protein Purification and Analysis

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LB broth was purchased from Tpi Research Products International Corporation. The HisTrap and gel filtration columns were obtained from GE Healthcare. Mini-PROTEAN precast gels were purchased from Bio-Rad. Bis-sulfosuccinimidyl suberate (BS3), immobilized pepsin resin and trypsin were purchased from Thermo Scientific. The C18 reverse phase column was purchased from Waters Corporation. D2O was obtained from Cambridge Isotope Laboratories. All other buffers, purification reagents, and chemicals used in this project were purchased from Sigma/Aldrich, unless otherwise noted.
Ren Z., Ranganathan S., Zinnel N.F., Russell W.K., Russell D.H, & Raushel F.M. (2015). Subunit Interactions within the Carbon-Phosphorus Lyase Complex from Escherichia coli. Biochemistry, 54(21), 3400-3411.
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6

Recombinant Expression of NiV V Protein

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The gene encoding the
NiV V protein (accession AAK50551) was codon-optimized for expression
in E. coli (GeneScript, NJ) prior to
subcloning into a modified pET15b plasmid containing a maltose-binding
protein (MBP) tag followed by a His6 tag. MDA5 and MDA5SF2 were amplified from human complementary DNA by polymerase
chain reaction. All sequences were confirmed with Sanger sequencing.
Recombinant proteins were expressed in E. coli BL21(DE3) cells at 18 °C for 16 h with 0.25 mM IPTG induction.
Cells were pelleted down by centrifugation and re-suspended in lysis
buffer containing 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, and protease
inhibitors prior to lysis with an EmulsiFlex-C5 homogenizer (Avestin,
Canada). Lysate was clarified by centrifugation at 60,000g and then used for pulldown assays or further protein purification
using a series of affinity, ion exchange, and gel filtration columns
(GE Healthcare, IL). The MBP-tag on MDA5SF2 was cleaved
using TEV protease and removed by affinity purification. Final protein
samples were assessed by SDS-PAGE.
Wagner N.D., Liu H., Rohrs H.W., Amarasinghe G.K., Gross M.L, & Leung D.W. (2021). Nipah Virus V Protein Binding Alters MDA5 Helicase Folding Dynamics. ACS Infectious Diseases, 8(1), 118-128.
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7

Ctr1 Cu(I) Transporter Peptide Characterization

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The C-terminal 13 amino acids of the human Ctr1 Cu(I)-transporter (Ctr1c), KKAVVVDITEHCH, were custom-ordered as a lyophilized peptide from Sigma. DTT (dithiotreitol), TCEP-HCl (tris(2-carboxyethyl)phosphine), and IPTG (isopropyl 1-thio-β-d-galactopyranoside) were purchased from GoldBio. Tris-base, mono- and dibasic sodium phosphate, sodium acetate, ammonium persulfate (APS), EDTA, β-mercaptoethanol (BME), and sodium chloride were acquired from Fisher. Nitroblue terazolium and bathocuproinedisulfonic acid (BCS) were purchased from Alfa Aesar. Primers for site-directed mutagenesis and zinc sulfate heptahydrate were bought from Sigma. Maleimide-polyethyleneglycol 2000 was purchased from nanocs. Tetramethylethylenediamine (TEMED) was purchased from Thermo Scientific, while riboflavin was acquired from Acros Organics. Alexa-488-succinimidyl ester was purchased from Life Technologies. Cu(I)-(CH3CN4)PF6 was purchased from Strem Chemicals. His-Trap Nickel affinity columns, SQ (anion exchange) columns, and gel filtration columns were purchased from GE LifeSciences.
Skopp A., Boyd S.D., Ullrich M.S., Liu L, & Winkler D.D. (2019). Copper–zinc superoxide dismutase (Sod1) activation terminates interaction between its copper chaperone (Ccs) and the cytosolic metal-binding domain of the copper importer Ctr1. Biometals, 32(4), 695-705.
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