Gel filtration calibration kit hmw

Manufactured by GE Healthcare

Sourced in United States, Sweden

The Gel Filtration Calibration Kit HMW is a laboratory equipment product designed for the calibration of gel filtration chromatography columns. The kit contains a set of high molecular weight protein standards that can be used to determine the molecular weight range and separation characteristics of the gel filtration column.

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Lab products found in correlation

Ne per nuclear extraction kit, by Thermo Fisher Scientific (5 mentions) Microcon centrifugal filter, by Merck Group (5 mentions) Superdex 200 10 300 gl column, by GE Healthcare (4 mentions) Superose 6 size exclusion column, by GE Healthcare (4 mentions) Superdex 200 column, by GE Healthcare (2 mentions) Kta purifier system, by GE Healthcare (2 mentions) Superose 6 10 300 gl column, by GE Healthcare (2 mentions) Akta system, by GE Healthcare (2 mentions) Ribonuclease, by ITW Reagents (2 mentions) Mini protean tgx gel, by Bio-Rad (2 mentions) Nanodrop, by Thermo Fisher Scientific (2 mentions) Superdex 200 hr 10 30 column, by Cytiva (1 mentions) Precast protein gel, by Bio-Rad (1 mentions) Superdex 200 increase 10 300 gl column, by GE Healthcare (1 mentions) 200 increase 10 300 gl column, by GE Healthcare (1 mentions) Superdex 200, by Cytiva (1 mentions) Superdex 200 increase, by GE Healthcare (1 mentions) Superose 6 10 300 gl, by GE Healthcare (1 mentions) Superose 6 gel filtration column, by GE Healthcare (1 mentions) Superdex 200 10 300 column, by GE Healthcare (1 mentions)

38 protocols using gel filtration calibration kit hmw

Determination of FadB' Molecular Mass

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The molecular mass of FadB’ was determined on a Superdex 200 HR 10/30 column (Amersham Pharmacia Biotech) at a flow rate of 1 ml/min in 50 mM sodium phosphate buffer (pH7.4) containing 150 mM NaCl. Molecular mass standards used for calibration were from the Gel Filtration Calibration Kit HMW (GE Healthcare, UK).

Volodina E, & Steinbüchel A. (2014). (S)-3-hydroxyacyl-CoA dehydrogenase/enoyl-CoA hydratase (FadB’) from fatty acid degradation operon of Ralstonia eutropha H16. AMB Express, 4, 69.

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Superdex 200 Size Exclusion Chromatography

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Peak fractions from a Source 15Q column were pooled and concentrated to app. 6 mg/mL using a Vivaspin 6 spin filter (Sartorius) with a 5 kDa molecular mass cut-off. During concentration, the buffer was exchanged to 20 mM Hepes, pH=7.5,150 mM NaCl, 5 mM BME. 400 μL of the protein sample was loaded on a Superdex 200 increase 10/300 GL column (GE Healthcare), which had been pre-equilibrated in the same buffer. The protein was eluted at 0.4 mL/min while measuring OD280. The elution volume (V_e) from the Superdex 200 increase 10/300 GL column was used to calculate the apparent molecular mass of the sample based on a calibration run with the Gel Filtration Calibration Kit HMW (GE Healthcare). Peak fractions from the analytical SEC run were concentrated to 4 mg/ml, and a reaction mixture containing 63 μL of VapXD complex and 7 μL of 0.5% or 1% glutaraldehyde was prepared. At the time points 5, 10, 20, 40, 60, and 120 min, 11 μL aliquots were removed, and the reaction quenched with 4 μL of 2 M Tris, pH=8.0. Samples were analysed on precast protein gels (Bio Rad).

Bertelsen M.B., Senissar M., Nielsen M.H., Bisiak F., Cunha M.V., Molinaro A.L., Daines D.A, & Brodersen D.E. (2020). Structural Basis for Toxin Inhibition in the VapXD Toxin-Antitoxin System. Structure (London, England : 1993), 29(2), 139-150.e3.

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Gel Filtration Chromatography of Protein-Ligand Complexes

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A Superdex™ 200 increase 10/300 GL column was run in buffer D (25 mM HEPES, 150 mM NaCl, 5 mM TCEP, pH 7.4) at a flow-rate of 0.5 mL/min and calibrated with the Gel Filtration Calibration Kit HMW (GE Healthcare). A linear fit a plot of K_av vs. log M_r gave R²= 0.99. 30 µM protein was incubated with ligands, e.g., Zn, GTP, and/or GDP, on ice for 1 h, and 100 µL sample injected.

Nairn B.L., Lonergan Z.R., Wang J., Braymer J.J., Zhang Y., Calcutt M.W., Lisher J.P., Gilston B.A., Chazin W.J., de Crécy-Lagard V., Giedroc D.P, & Skaar E.P. (2016). The response of Acinetobacter baumannii to Zinc starvation. Cell host & microbe, 19(6), 826-836.

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Characterization of RibBX-FMN Complex

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A Superdex 200 10/300 GL column (GE Healthcare) was equilibrated and chromatographed in 50 mM Tris, 150 mM NaCl, 10 mM MgCl₂, 2 mM TCEP, pH 7.5 at a flow-rate of 0.5 mL/min and calibrated with the Gel Filtration Calibration Kit HMW (GE Healthcare). A linear fit a plot of K_av vs. log M_r gave R²= 0.90. 100 μM RibBX was pre-incubated with 1.25 eq. FMN at 4 °C for 1 h. 100 μL samples were injected for each chromatography run.

Wang J., Lonergan Z.R., Gonzalez-Gutierrez G., Nairn B.L., Maxwell C.N., Zhang Y., Andreini C., Karty J.A., Chazin W.J., Trinidad J.C., Skaar E.P, & Giedroc D.P. (2019). Multi-metal restriction by calprotectin impacts de novo flavin biosynthesis in Acinetobacter baumannii. Cell chemical biology, 26(5), 745-755.e7.

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Protein Purification by Gel Filtration

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The gel filtration column (Superdex 200; Amersham Biosciences) was washed and equilibrated by cold PBS (4 °C). Proteins were passed over the gel filtration column. The flow speed rate was 0.4 mL min⁻¹. Fractions were collected every 0.5 mL per tube and analyzed by western blot. Molecular mass was determined by Gel Filtration Calibration Kit HMW (GE Healthcare).

Li Y., Yao C.F., Xu F.J., Qu Y.Y., Li J.T., Lin Y., Cao Z.L., Lin P.C., Xu W., Zhao S.M, & Zhao J.Y. (2019). APC/CCDH1 synchronizes ribose-5-phosphate levels and DNA synthesis to cell cycle progression. Nature Communications, 10, 2502.

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Gel Filtration of H2O2-Treated Cell Extracts

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Cells were treated with or without 10 μM H₂O₂ for 6 h and lysed by NP-40 buffer containing protease inhibitors (as above) for 30 min and centrifuged for 15 min at 13,000 rpm to remove cell debris. The gel filtration column (Superdex 200 Increase; GE Healthcare) was washed and equilibrated by cold PBS. Extracts were passed over the gel filtration column. The speed rate of flow is 0.4 ml/min. Fractions were collected every 0.25 ml per tube and analyzed by western blotting. Molecular mass was determined by Gel Filtration Calibration Kit HMW (GE Healthcare).

Liu Y., Guo J.Z., Liu Y., Wang K., Ding W., Wang H., Liu X., Zhou S., Lu X.C., Yang H.B., Xu C., Gao W., Zhou L., Wang Y.P., Hu W., Wei Y., Huang C, & Lei Q.Y. (2018). Nuclear lactate dehydrogenase A senses ROS to produce α-hydroxybutyrate for HPV-induced cervical tumor growth. Nature Communications, 9, 4429.

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Estimating Protein-Detergent Complex Size

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The
hydrodynamic sizes of protein–detergent complexes can be estimated
by using size exclusion chromatography on appropriate columns. Pure
AqpZ (1 mg) was injected in 500 μL on a Superose 6 10/300 GL
(GE Healthcare) column previously equilibrated with elution buffer
(NaCl 200 mM, KCl 2.7 mM, K₂HPO₄ 1.4 mM, Na₂HPO₄ 16.29 mM, 0.05% DDM, pH 8.0). The column was
developed with the same buffer.
To determine the volumes of
protein–detergent complexes, the Superose 6 10/300 GL column
was calibrated, using the Gel Filtration Calibration Kit HMW (GE Healthcare)
supplemented with lysozyme (14 kDa) and Atto532 in elution buffer.
The partition coefficient, K_d, for each
protein was calculated from K_d = (V_m – V_o)/(V_t – V_o),
where V_o is the void volume determined
with blue dextran, V_t is the geometric
bed volume determined with Atto532, and V_m is the elution volume at the peak monitored by absorbance at 280
nm. The volumes of the proteins in the calibration kit were estimated
by assuming a protein density of 1.212 × 10^–3 nm³ Da^–1.^{55 (link)}

Schmidt V, & Sturgis J.N. (2017). Making Monomeric Aquaporin Z by Disrupting the Hydrophobic Tetramer Interface. ACS Omega, 2(6), 3017-3027.

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Protein-Zn(II) Interaction Analysis

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A Superdex 300 (Sepax SRT-10, 10 × 300 mm, 10 μm particle size) GL column was run in buffer [25 mM HEPES, 150 mM NaCl, and 5 mM TCEP (pH 7.4)] at a flow rate of 0.5 mL min⁻¹ and calibrated with the Gel Filtration Calibration Kit HMW (GE Healthcare). A linear-fit plot of K_av versus log M_r gave R² = 0.99. Varying concentrations of protein (5–100 μM) were incubated with Zn^II on ice for 1 h, and 100 μL samples were injected onto a preequilibrated column.

Jordan M.R., Wang J., Weiss A., Skaar E.P., Capdevila D.A, & Giedroc D.P. (2019). Mechanistic Insights into the Metal-Dependent Activation of ZnII-Dependent Metallochaperones. Inorganic chemistry, 58(20), 13661-13672.

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Protein Size Determination Methods

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For SEC assays, the purified protein samples were loaded onto a Superdex 200 column (GE Healthcare) equilibrated with 25 mM Tris–HCl pH 8.0 containing 100 mM or 500 mM NaCl. For the column calibration, besides Ec_RND (45.3 kDa) and Tle1 (96.5 kDa), conalbumin (75 kDa) and aldolase 1 (158 kDa) from the Gel Filtration Calibration Kit HMW (GE Healthcare) were used. The experimental data, with the exception of that for At_NrnC, were used for the linear fit.
Analytical ultracentrifugation experiments were performed using an XL-I analytical ultracentrifuge (Beckman Coulter, Fullerton, CA, United States) equipped with a four-cell An-60 Ti rotor. At_NrnC was diluted to 1 mg/mL in 25 mM Tris–HCl pH 8.0 buffer containing 100 mM NaCl. To determine the influence of metal binding, 5 mM MnCl₂ was added. The corresponding buffer was used as the reference solution. All samples were centrifuged at 60,000 rpm at 20°C. Analysis of the sedimentation velocity results was performed using the programme Sedfit (Brown and Schuck, 2006 (link)).

Yuan Z., Gao F., Yin K, & Gu L. (2019). NrnC, an RNase D-Like Protein From Agrobacterium, Is a Novel Octameric Nuclease That Specifically Degrades dsDNA but Leaves dsRNA Intact. Frontiers in Microbiology, 9, 3230.

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Estimating the Size of PfGCN5 Complex

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To estimate the size of the PfGCN5 complex, nuclear extract from the PfGCN5::PTP was incubated with IgG beads, eluted by TEV protease cleavage as described above, and applied to a Superose 6 gel filtration column (GE Healthcare). Molecular mass standards (Gel Filtration Calibration Kit HMW, GE Healthcare) were run under the same conditions to estimate the size of the complex. The fractions were analyzed by Western blotting using antibodies against the PTP tag, PfPHD1 and PfPHD2, while HAT activity in the fractions was measured as described previously [57 (link)].

Miao J., Wang C., Lucky A.B., Liang X., Min H., Adapa S.R., Jiang R., Kim K, & Cui L. (2021). A unique GCN5 histone acetyltransferase complex controls erythrocyte invasion and virulence in the malaria parasite Plasmodium falciparum. PLoS Pathogens, 17(8), e1009351.

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