Econo pac 10dg

Manufactured by Bio-Rad

Sourced in United States

The Econo-Pac 10DG is a desalting column designed for the rapid and efficient removal of low molecular weight compounds from protein solutions. It is a pre-packed column that is simple to use and requires no column packing or preparation. The Econo-Pac 10DG utilizes a dextran-based gel filtration matrix to achieve desalting and buffer exchange.

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Lab products found in correlation

Histrap ff crude column, by GE Healthcare (1 mentions) Isopropylthio β d galactoside, by Merck Group (1 mentions) Pro k software, by Applied Photophysics (1 mentions) Diode array detector, by Applied Photophysics (1 mentions) Sx20 stopped flow spectrophotometer, by Applied Photophysics (1 mentions) Uv 2501pc scanning spectrophotometer, by Shimadzu (1 mentions) Pet15b, by Merck Group (1 mentions) Bl21 de3 plys, by New England Biolabs (1 mentions) Amicon ultra centrifugal filter, by Merck Group (1 mentions) Fetal calf serum (fcs), by Merck Group (1 mentions) V 660 spectrophotometer, by Jasco (1 mentions) Cary 300, by Agilent Technologies (1 mentions) Simvastatin, by Merck Group (1 mentions) Dansyl chloride, by Thermo Fisher Scientific (1 mentions) Amicon ultra 0.5 ml centrifugal filter, by Merck Group (1 mentions)

Spelling variants of this product

Econo-Pac 10DG desalting column (35 citations) Econo-Pac 10DG column (21 citations) Econo-Pac (7 citations)

17 protocols using econo pac 10dg

Recombinant PPX Mutant Purification

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Ultracompetent E. coli were transformed with pTrcHisB vectors coding the various PPX deletion mutants. Recombinant protein expression was induced by 0.5 mM isopropylthio-β-D-galactoside (Sigma-Aldrich) at 37 °C for 4 h. Bacteria were collected by centrifugation, resuspended in binding buffer (20 mM NaH₂PO₄, 500 mM NaCl and 20 mM imidazole, pH 7.4) and lysed by sonication. Cell lysates were centrifuged (10,000 g for 10 min at 4 °C) and supernatants were loaded on 1 ml HisTrap FF crude column (GE Healthcare). Following washing, bound proteins were eluted with 20 mM NaH₂PO₄, 500 mM NaCl and 500 mM imidazole, pH 7.4. Fractions containing mutants were combined, and solvent was changed to PBS, pH 7.4, using desalting columns (Econo-Pac 10 DG, Bio-Rad). Protein concentrations were determined by the Bradford method. Coomassie brilliant blue staining assessed protein purity. Western blotting was performed using 6xHis-tag antibody (1:1,000, Merck Millipore, catalogue number #70796-3) and horseradish peroxidase (HRP)-coupled anti-mouse Fc antibody (1:5,000, Jackson ImmunoResearch, #115-035-003). Full scans of the western blots are shown in Supplementary Fig. 4.

Labberton L., Kenne E., Long A.T., Nickel K.F., Di Gennaro A., Rigg R.A., Hernandez J.S., Butler L., Maas C., Stavrou E.X, & Renné T. (2016). Neutralizing blood-borne polyphosphate in vivo provides safe thromboprotection. Nature Communications, 7, 12616.

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Measuring Oxygen Dissociation from Ngb Mutant

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The rate of oxygen dissociation from Ngb-H64Q-CCC was determined by ligand replacement. The experiments were carried out at 25 °C in an Applied Photophysics SX-20 stopped-flow spectrophotometer with a diode array detector (Applied Photophysics Ltd.) contained in an anaerobic glove box (Coy Laboratory Products). 100 mM sodium phosphate buffer, pH 7.4 was used for all the solutions. The Ngb samples were reduced by excess sodium dithionite in the glove box and the deoxy-Ngb was run through a gravity size-exclusion column (Econopac 10DG, BioRad) to remove the excess of reductant. The protein was mixed with air-saturated buffer ([O₂] ≈ 260μM) in 1:4 (v/v) or 1.5:1 (v/v) ratios to form quantitatively the Ngb-oxy complex and achieve final oxygen concentrations of ≈ 208 μM (1:4) or ≈ 104 μM (1.5/1). This sample was then mixed different concentrations of CO buffer made by mixing CO-saturated buffer ([CO] ≈ 1 mM) and anaerobic buffer in the stopped-flow instrument. The spectrum in the visible range (350–730 nm) was sampled every 1.24 ms for a reaction time of 1 s. Spectral changes were consistent with a decay of the Ngb-oxy complex to form the Ngb-CO species. Traces over the whole spectral range (300–730nm) where fitted simultaneously for each experiment using the Pro-K software (Applied Photophysics Ltd.) to calculate the observed oxygen dissociation rate.

Azarov I., Wang L., Rose J.J., Xu Q., Huang X.N., Belanger A., Wang Y., Guo L., Liu C., Ucer K.B., McTiernan C.F., O’Donnell C.P., Shiva S., Tejero J., Kim-Shapiro D.B, & Gladwin M.T. (2016). Five-coordinate H64Q neuroglobin as a ligand-trap antidote for carbon monoxide poisoning. Science translational medicine, 8(368), 368ra173.

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Anaerobic Protein Spectroscopic Measurement

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All reagents were of analytical grade and
were purchased from Sigma-Aldrich, apart from NADH, which was purchased
from Melford (Ipswich, UK). All spectroscopic measurements were performed
in 50 mM potassium phosphate buffer (pH 7.0). For anaerobic measurements,
all buffers were degassed with N₂ before being introduced
into the anaerobic glovebox (<5 ppm of O₂). Prior to
performing anaerobic spectroscopic measurements, protein samples were
taken into the glovebox, oxidized with a few grains of potassium ferricyanide,
and passed down a Bio-Rad Econo-Pac 10DG-desalting column equilibrated
with anaerobic buffer.

Hedison T.M., Shenoy R.T., Iorgu A.I., Heyes D.J., Fisher K., Wright G.S., Hay S., Eady R.R., Antonyuk S.V., Hasnain S.S, & Scrutton N.S. (2019). Unexpected Roles of a Tether Harboring a Tyrosine Gatekeeper Residue in Modular Nitrite Reductase Catalysis. ACS Catalysis, 9(7), 6087-6099.

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Enzymatic Characterization of FDTS

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The buffer of stock solutions of FDTS was removed by passing through a desalting column (Econo-Pac 10DG, Bio-Rad) equilibrated with a solution of 50 mM NaCl whose pH had been adjusted to 8.0 using HCl and/or NaOH. Enzyme was diluted into the same solvent to a concentration of 10 μM FAD, and phenol red was added (20 μM) from a concentrated stock solution in 50 mM NaCl whose pH had been adjusted to 8.0. The pH of the enzyme-dye mixture was checked at the start of an experiment and adjusted to 8.0 with HCl or NaOH if deviations were detected. Titrations were performed on a Shimadzu UV-2501PC scanning spectrophotometer at 25 °C. Small aliquots were added of a concentrated stock solution of dUMP made in 50 mM NaCl, pH 8.0, and absorbance spectra were recorded.

Stull F.W., Bernard S.M., Sapra A., Smith J.L., Zuiderweg E.R, & Palfey B.A. (2016). Deprotonations in the Reaction of Flavin-Dependent Thymidylate Synthase. Biochemistry, 55(23), 3261-3269.

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Purification and Oxidation of Human CPR

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Homo sapiens cytochrome P450 reductase (CPR), lacking the 60 amino acid membrane‐binding N‐terminus, was expressed from plasmid pET15b (Millipore, Watford, Hertfordshire, UK) in Escherichia coli BL21 (DE3) pLys (New England BioLabs, Hitchin, Hertfordshire, UK), and purified by DEAE–Sepharose anion exchange chromatography followed by nickel–Sepharose affinity chromatography, as described previously 40, 41. Prior to use, CPR was oxidized using a few grains of potassium ferricyanide, and immediately passed through an Econo‐Pac 10DG desalting column (Bio‐Rad, Hemel Hempstead, UK) to remove surplus oxidant. The oxidized CPR concentration was determined by absorption spectroscopy using an absorption coefficient (ε) of 22 mm⁻¹·cm⁻¹ at 454 nm 22.

Hedison T.M., Hay S, & Scrutton N.S. (2015). Real‐time analysis of conformational control in electron transfer reactions of human cytochrome P450 reductase with cytochrome c. The Febs Journal, 282(22), 4357-4375.

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Purification of NEM-conjugated Hemoglobin

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NEM (Thermo Fisher Scientific, ≥99%) was incubated with hemoglobin, isolated, and purified as described above in a 3:1 ratio of NEM/heme (i.e., in excess of β-Cys93 sites) with gentle rocking (Labnet ProBlot Rocker 25 XLD) for 1 hour at room temperature. Excess NEM was removed by a gravity-flow chromatography column (Econo-Pac 10DG; Bio-Rad) preequilibrated with NS. Then the NEMHb was concentrated by differential ultracentrifugation (Amicon Ultra centrifugal filters, 50 kDa molecular weight cutoff; MilliporeSigma), aliquoted, and stored at –80°C. As above, holoprotein concentration and species fractionation were assessed using UV-Vis spectral deconvolution, and only preparations that yielded > 99% Fe²⁺-O₂ hemoglobin were stored for further use.

Xu Q., Rose J.J., Chen X., Wang L., DeMartino A.W., Dent M.R., Tiwari S., Bocian K., Huang X.N., Tong Q., McTiernan C.F., Guo L., Alipour E., Jones T.C., Ucer K.B., Kim-Shapiro D.B., Tejero J, & Gladwin M.T. (2022). Cell-free and alkylated hemoproteins improve survival in mouse models of carbon monoxide poisoning. JCI Insight, 7(21), e153296.

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Isolation and Oxidation of LDL Lipoprotein

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LDL (relative density 1.019 to 1.063 g/ml) was isolated from human plasma by sequential ultracentrifugation and was flotated through a potassium bromide solution of d=1.063 g/ml. After centrifugation LDL was filtered (0.45 µm pore size) and used immediately for further modification. LDL was transferred into PBS containing 100 µM EDTA by gel filtration through a size exclusion column (Bio-Rad Econo-Pac 10DG). The preparation of hypochlorite oxidized LDL was performed as described previously (Arnhold et al., 1991[3 (link)]) with minor modifications. The molar excess of hypochlorite over LDL was in the range of 400-600. After allowing the hypochlorite reagent to react with LDL for 15 min on ice, LDL was again passed over a size exclusion column (Bio-Rad 10DG) equilibrated with PBS containing 100 µM EDTA to remove excess reagent. Agarose gel electrophoresis of hypochlorite (HOCl) ox-LDL particles revealed an about twofold increase of the relative electrophoretic mobility. For the preparation of lipoprotein deficient serum (LPDS) fetal calf serum (Sigma) was depleted of lipoproteins by flotation at 100 000g with a density of 1.21 g/ml for 48 hours. After flotation the lipoprotein deficient serum was excessively dialyzed against saline and sterile filtered (0.22 µm pore size).

Brunner M., Gruber M., Schmid D., Baran H, & Moeslinger T. (2015). Proliferation of macrophages due to the inhibition of inducible nitric oxide synthesis by oxidized low-density lipoproteins. EXCLI Journal, 14, 439-451.

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Fluorene-NHS Conjugation with c(RGDfK)

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Fluorene-NHS 2 (20 mg, 0.010 mmol) and c(RGDfK) (5 mg, 0.008 mmol) were dissolved in DMF (1.5 mL), and the solution stirred for 24 h under nitrogen at room temperature. Solvent was removed under vacuum, and crude product was then passed through a size exclusion column (12 cm length, Bio-Rad Econo-Pac 10DG, MWCO) in pure water to allow collection of pure RGD conjugate fractions, yielding a red oil (15 mg, 60% yield). HRMS-MALDI-DTL: High resolution MS shows a series of ions centered around m/z 2540 [C₁₀₂H₁₀₅N₁₃O₁₁(C₂H₄O)₂₀]⁺ which are fragments related to the various ethoxylate oligomers that differ by Δm/z 44, an ethoxy unit.

Yue X., Morales A.R., Githaiga G.W., Woodward A.W., Tang S., Sawada J., Komatsu M., Liu X, & Belfield K.D. (2015). RGD-conjugated Two-photon Absorbing Near-IR Emitting Fluorescent Probes for Tumor Vasculature Imaging. Organic & biomolecular chemistry, 13(43), 10716-10725.

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LDL Oxidation Kinetics Assay

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EDTA and salt from the density gradient were removed from the LDL solution using a prepacked column (Econo-Pac 10DG; Bio-Rad, Richmond, CA, USA), as described by Puhl et al.²⁰ (link) The concentration of LDL, free of EDTA, was adjusted to 50 μg/mL of protein with 10mM phosphate-buffered saline (pH 7.4), and it was transferred into a quartz cell for spectrophotometric analysis. An aliquot of the sample (100 μL) was then added to the cell. Oxidation was started at 37°C by the addition of AAPH to a final concentration of 1mM. Trolox, as control, was dissolved in ethanol, dried under nitrogen, and incubated with LDL solution (final Trolox concentrations of 0.625 μg/mL, 1.25 μg/mL, and 2.5 μg/mL). The kinetics of LDL oxidation was determined from the change in conjugated diene formation by monitoring the change in absorbance at 234 nm using a Shimadzu UV-3100 spectrophotometer (UVeVISeNIR scanning; Shimadzu Corp., Kyoto, Japan). Absorbance was recorded every 10 minutes for 4 hours. The changes in absorbance at 234 nm and time were divided into three phases: lag, propagation, and decomposition.

Lin H.H., Charles A.L., Hsieh C.W., Lee Y.C, & Ciou J.Y. (2014). Antioxidant effects of 14 Chinese traditional medicinal herbs against human low-density lipoprotein oxidation. Journal of Traditional and Complementary Medicine, 5(1), 51-55.

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Inward-locked SLC26Dg Protein Expression and Purification

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SLC26Dg-IL (I45C/A142C, inward-locked) was expressed and purified as described method for cysteine variants used in PELDOR studies. The eluted and HRV 3 C protease-cleaved protein was subjected to SEC in buffer C supplemented with 3 mm DTT. Proteins from peak fractions were pooled and DTT was removed with an Econo-Pac 10DG desalting column (Bio-rad) which was pre-equilibrated with buffer C. The concentration of protein was adjusted to ~ 7 µm and oxidative cross-linking was initiated by adding a 10-fold concentrated CuPhen stock (3 mm CuSO₄, 9 mm 1,10-phenanthroline monohydrate, freshly prepared) into the protein solution. The sample was incubated at room temperature for 45 min with gentle agitation and subsequently 0.5 m Na-EDTA, pH 7.0 was added to a final concentration of 20 mm to quench the reaction. The locked protein was injected for SEC to remove the cross-linking reagent and peak fractions were pooled and used for subsequent reconstitution and transport studies.

Chang Y.N., Jaumann E.A., Reichel K., Hartmann J., Oliver D., Hummer G., Joseph B, & Geertsma E.R. (2019). Structural basis for functional interactions in dimers of SLC26 transporters. Nature Communications, 10, 2032.

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