Bovine thyroglobulin, by Bio-Rad - Product details

1

Characterization of CsgG Protein Structure

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Example 3

About 0.5 mg each of detergent-solubilized CsgG (0.5% C8E4, 4 mM LOAO) and CsgGc₁s were applied to a Superdex 200 10/300 GL analytical gel filtration column (GE Healthcare) equilibrated with 25 mM Tris-HCl pH 8.0, 500 mM NaCl, 1 mM OTT, 4 mM LOAO and 0.5% C8E4 (CsgG) or with 25 mM Tris-HCl pH 8.0, 200 mM NaCl (CsgGc₁s), and run at 0.7 ml min-¹The column elution volumes were calibrated with bovine thyroglobulin, bovine γ-globulin, chicken ovalbumin, horse myoglobulin and vitamin 8₁₂(Bio-Rad) (FIG. 7). Membrane-extracted CsgG, 20 μg of the detergent-solubilized protein was also run on 3-10% blue native PAGE using the procedure described in Swamy, M., et at, Sci. STKE 2006, pl4, [dx.doi.org/10.1126/stke.3452006pl4(2006)] (FIG. 7). NativeMark (Life Technologies) unstained protein standard (7 μl) was used for molecular mass estimation. Mature CsgG is predominantly found as discrete nonameric poreforming particles with C9 symmetry, as well as tail-to-tail dimers of nonameric pores (i.e. octadecamers with 09 symmetry). For the purpose of nanopore sensing applications, a preferred state of the proteins is a single nonameric pore. The population of nonameric versus 09 octadecameric pores can be increased by heathing samples prior to size exclusion chromatography and/or insertion in a lipid bilayer for nanopore sensing applications.

US11034734B2. Mutant pores (2021-06-15). Oxford Nanopore Technologies Ltd. [GB], VIB VZW [BE], Vrije Universiteit Brussel [BE]. Inventors: Stefan Howorka [GB], Han Remaut [BE], Lakmal Jayasinghe [GB], Elizabeth Jayne Wallace [GB], James Anthony Clarke [GB], Richard George Hambley [GB], Jonathan Bankes Pugh [GB].

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2

Molecular Weight Determination of SaTrmK

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Size-exclusion chromatography was carried out at 20 °C on a Superdex 200 10/300 Gl column pre-equilibrated with 20 mM Hepes pH 7.5, using an AKTA Purifier FPLC system (GE Healthcare). Samples (1 ml) were loaded onto the column at 1 mg/ml. Vitamin B12 (1350 Da), horse myoglobin (17,000 Da), chicken ovalbumin (44,000 Da), bovine γ-globulin (15,8000 Da), and bovine thyroglobulin (670,000 Da) (Bio-Rad) were used as MW standards. The logarithm of the MW of the standards were plotted against the ratios of the respective elution volumes (v_e) to the void volume (v₀). The points were then fitted to a linear regression, and the values of slope and intercept were used to determine the molecular weight of SaTrmK in solution.

Sweeney P., Galliford A., Kumar A., Raju D., Krishna N.B., Sutherland E., Leo C.J., Fisher G., Lalitha R., Muthuraj L., Sigamani G., Oehler V., Synowsky S., Shirran S.L., Gloster T.M., Czekster C.M., Kumar P, & da Silva R.G. (2022). Structure, dynamics, and molecular inhibition of the Staphylococcus aureus m1A22-tRNA methyltransferase TrmK. The Journal of Biological Chemistry, 298(6), 102040.

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3

Protein Size Fractionation by SEC

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0.5 ml of 2 mg/ml cell extract was 0.22-μm filtered and resolved on Superdex 200 10/300 GL preparative grade column (GE Healthcare) in 50 mM Tris–HCl, pH 7.4, 1 mM EDTA, 0.1 M sodium chloride, 0.03% Brij-35, and 2 mM DTT at 0.4 ml/min flow. 0.5 ml fractions were collected. Molecular weight markers (Bio-Rad) were used: bovine thyroglobulin (670 kD), bovine gamma globulin (158 kD), and chicken ovalbumin (44 kD).

Inesta-Vaquera F., Chaugule V.K., Galloway A., Chandler L., Rojas-Fernandez A., Weidlich S., Peggie M, & Cowling V.H. (2018). DHX15 regulates CMTR1-dependent gene expression and cell proliferation. Life Science Alliance, 1(3), e201800092.

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4

Analytical Gel Filtration of CsgG Proteins

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About 0.5 mg each of detergent-solubilized CsgG (0.5% C8E4, 4 mM LDAO) and CsgG_C1S were applied to a Superdex 200 10/300 GL analytical gel filtration column (GE Healthcare) equilibrated with 25 mM Tris-HCl pH 8.0, 500 mM NaCl, 1 mM DTT, 4 mM LDAO and 0.5% C8E4 (CsgG) or with 25 mM Tris-HCl pH 8.0, 200 mM NaCl (CsgG_C1S), and run at 0.7 ml min⁻¹. The column elution volumes were calibrated with bovine thyroglobulin, bovine γ-globulin, chicken ovalbumin, horse myoglobulin and vitamin B₁₂ (Bio-Rad) (Extended Data Fig. 2). Membrane-extracted CsgG, 20 μg of the detergent-solubilized protein was also run on 3–10% blue native PAGE using the procedure described in ref. 31 (link) (Extended Data Fig. 2). NativeMark (Life Technologies) unstained protein standard (7 μl) was used for molecular mass estimation.

Goyal P., Krasteva P.V., Van Gerven N., Gubellini F., Van den Broeck I., Troupiotis-Tsaïlaki A., Jonckheere W., Péhau-Arnaudet G., Pinkner J.S., Chapman M.R., Hultgren S.J., Howorka S., Fronzes R, & Remaut H. (2014). Structural and mechanistic insights into the bacterial amyloid secretion channel CsgG. Nature, 516(7530), 250-253.

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5

Protein Size Analysis by SEC-HPLC

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Size-exclusion chromatography was performed by analytical HPLC (Jasco LC-2000 Plus) equipped with a PU 2880 Plus injector and a PDA MD 2018 detector (Jasco). The samples (50 μM in 100 mM Tris-HCl at pH 7.4) were separated by a system containing a Phenomenex BioSep-SEC-S3000 column (7.8 × 300 mm, 5 μm, resolution range of 15 to 2000 kDa, Phenomenex, Inc., Torrance, California, USA) using a flow rate of 1.0 mL/min in 100 mM Tris-HCl buffer (pH 7.4) and 50 mM NaCl. The elution profile was monitored by absorbance (λ = 280 nm). Bovine thyroglobulin (670 kDa), bovine gamma globulin (158 kDa), ovalbumin (44 kDa), myoglobin (17 kDa) and vitamin B₁₂ (1.35 kDa) were used as molecular weight standards (Bio-Rad). The chromatograms were analyzed using Jasco BORWIN, version 1.50, software (Jasco). The enzymes were reduced or oxidized by treatment with 5 mM TCEP or 1.2 molar excess of H₂O₂, respectively, for 30 min at 25 °C.

Tairum C.A., Santos M.C., Breyer C.A., Geyer R.R., Nieves C.J., Portillo-Ledesma S., Ferrer-Sueta G., Toledo JC J.r., Toyama M.H., Augusto O., Netto L.E, & de Oliveira M.A. (2016). Catalytic Thr or Ser Residue Modulates Structural Switches in 2-Cys Peroxiredoxin by Distinct Mechanisms. Scientific Reports, 6, 33133.

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6

CsgG Oligomerization and Nanopore Sensing

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Example 3

About 0.5 mg each of detergent-solubilized CsgG (0.5% C8E4, 4 mM LDAO) and CsgG_C1Swere applied to a Superdex 200 10/300 GL analytical gel filtration column (GE Healthcare) equilibrated with 25 mM Tris-HCl pH 8.0. 500 mM NaCl, 1 mM DTT, 4 mM LDAO and 0.5% C8E4 (CsgG) or with 25 mM Tris-HCl pH 8.0, 200 mM NaCl (CsgG_C1S), and run at 0.7 ml min⁻¹. The column elution volumes were calibrated with bovine thyroglobulin, bovine γ-globulin, chicken ovalbumin, horse myoglobulin and vitamin B₁₂(Bio -Rad) (FIG. 7). Membrane-extracted CsgG, 20 μg of the detergent-solubilized protein was also run on 3-10% blue native PAGE using the procedure described in Swamy, M., et al., Sci. STKE 2006, pl4, [http://dx.doi.org/10.1126/stke.3452006pl4(2006)] (FIG. 7). NativeMark (Life Technologies) unstained protein standard (7 μl) was used for molecular mass estimation. Mature CsgG is predominantly found as discrete nonarneric poreforrning particles with C9 symmetry, as well as tail-to-tail dimers of nonarneric pores (i.e. octadecamers with D9 symmetry). For the purpose of nanopore sensing applications, a preferred state of the proteins is a single nonameric pore. The population of nonameric versus D9 octadecameric pores can be increased by heathing samples prior to size exclusion chromatography and/or insetion in a lipid bilayer for nanopore sensing applications.

US10400014B2. Mutant CsgG pores (2019-09-03). Oxford Nanopore Technologies Ltd. [GB], VIB VZW [BE], Vrije Universiteit Brussel [BE]. Inventors: Stefan Howorka [GB], Han Remaut [BE], Lakmal Jayasinghe [GB], Elizabeth Jayne Wallace [GB], James Anthony Clarke [GB], Richard George Hambley [GB], Jonathan Bankes Pugh [GB].

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7

Protein Fractionation and Characterization

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Cells were lysed 72 hours post-transfection in Lysis Buffer (20 mM Tris-HCl pH 7.4, 137 mM NaCl, 2 mM EDTA, 25 mM β-glycerophosphate, 10% v/v glycerol, 1% Triton-X-100, protease and phosphatase inhibitors). Insoluble material was eliminated by centrifugation at 13200 rpm 20 minutes in an Eppendorf 5415R centrifuge. Soluble material was quantified and 3 mg of protein were fractionated by HPLC in a Superose 12 10/300 GL column (Pharmacia-GE Healthcare). Fractions were eluted in Elution Buffer (50 mM Tris HCl, pH 7.5, 1 mM EDTA, 100 mM KCl, sodium azide 0.025%), previously filtered (0.22 μm) and degassed, at a flow rate of 0.4 ml/min and monitored at 280 nm. Fractions of 0.2 ml were collected, precipitated and resolved on polyacrylamide gels and the following molecular weight markers (BioRad) were used: bovine thyroglobulin (670 kDa), bovine γ-globulin (158 kDa), chicken ovalbumin (44 kDa), horse myoglobin (17 kDa) and vitamin B12 (1.35 kDa).

Cantarero L., Sanz-García M., Vinograd-Byk H., Renbaum P., Levy-Lahad E, & Lazo P.A. (2015). VRK1 regulates Cajal body dynamics and protects coilin from proteasomal degradation in cell cycle. Scientific Reports, 5, 10543.

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8

Protein Purification by Size Exclusion Chromatography

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0.5 ml of 2 mg/ml cell extract was 0.22-μm filtered and resolved on Superdex 200 10/300 GL preparative grade column (GE Healthcare) in 50 mM Tris–HCl, pH 7.4, 1 mM EDTA, 0.1 M sodium chloride, 0.03% Brij-35, and 2 mM DTT at 0.4 ml/min flow. 0.5 ml fractions were collected. Molecular weight markers (Bio-Rad) were used: bovine thyroglobulin (670 kD), bovine gamma globulin (158 kD), and chicken ovalbumin (44 kD).

Inesta-Vaquera F., Chaugule V.K., Galloway A., Chandler L., Rojas-Fernandez A., Weidlich S., Peggie M, & Cowling V.H. (2018). DHX15 regulates CMTR1-dependent gene expression and cell proliferation. Life Science Alliance, 1(3), e201800092.

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9

Size-Exclusion Chromatography of MtL

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The homogeneity of MtL in solution was further probed by analytical size-exclusion chromatography on a Superdex 200 HR 10/30 column (Pharmacia). The column was equilibrated with 50 mM NaH₂PO₄, 150 mM NaCl, pH 7 and calibrated using a set of molecular weight protein standards (Bio-Rad) comprising bovine thyroglobulin (670 kDa), bovine gamma globulin (158 kDa), chicken ovalbumin (44 kDa) and horse myoglobulin (17 kDa). The MtL sample and the standards were all eluted at 0.4 ml/min.

Ernst H.A., Jørgensen L.J., Bukh C., Piontek K., Plattner D.A., Østergaard L.H., Larsen S, & Bjerrum M.J. (2018). A comparative structural analysis of the surface properties of asco-laccases. PLoS ONE, 13(11), e0206589.

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10

Size-Exclusion Chromatography of rPbPrx1

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Size-exclusion chromatography experiments were performed by analytical HPLC equipped with a PU 2880 Plus injector and a PDA MD 2018 detector (LC-2000 series; Jasco, Tokyo, Japan). The samples (50 μM in 100 mM Tris-HCl, pH 7.4) were separated by a system containing a Phenomenex BioSep-SEC-S3000 column (7.8 × 300 mm, 5 μm, resolution range of 1–300 kDa, Phenomenex, Inc., Torrance, California, USA) at a flow rate of 1.0 mL min⁻¹ in 100 mM Tris-HCl, pH 7.4, containing 50 mM NaCl. The elution profile was monitored by absorbance at λ = 280 nm. Bovine thyroglobulin (670 kDa), bovine gamma globulin (158 kDa), ovalbumin (44 kDa), myoglobin (17 kDa), and vitamin B₁₂ (1.35 kDa) were used as molecular standards (Bio-Rad Laboratories, Richmond, USA). Chromatograms were analyzed using Jasco BORWIN, version 1.50, software (Jasco, Tokyo, Japan). The redox treatments for rPbPrx1 were either 5 mM TCEP (reductant) or 1.2 molar equivalent of hydrogen peroxide (oxidant) for 30 min, at 25°C prior to the chromatographic runs.

Longo L.V., Breyer C.A., Novaes G.M., Gegembauer G., Leitão NP J.r., Octaviano C.E., Toyama M.H., de Oliveira M.A, & Puccia R. (2020). The Human Pathogen Paracoccidioides brasiliensis Has a Unique 1-Cys Peroxiredoxin That Localizes Both Intracellularly and at the Cell Surface. Frontiers in Cellular and Infection Microbiology, 10, 394.

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Bovine thyroglobulin

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12 protocols using bovine thyroglobulin

Characterization of CsgG Protein Structure

Molecular Weight Determination of SaTrmK

Protein Size Fractionation by SEC

Analytical Gel Filtration of CsgG Proteins

Protein Size Analysis by SEC-HPLC

CsgG Oligomerization and Nanopore Sensing

Protein Fractionation and Characterization

Protein Purification by Size Exclusion Chromatography

Size-Exclusion Chromatography of MtL

Size-Exclusion Chromatography of rPbPrx1

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