Np00061

Manufactured by Merck Group

The NP00061 is a laboratory instrument designed for general scientific applications. It is a versatile device that can perform a variety of tasks essential for research and analysis. The core function of this product is to provide reliable and accurate measurements to support various experimental and investigative processes. Further details on the intended use or specific applications of this equipment are not available.

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Lab products found in correlation

3 protocols using np00061

Western Blot Analysis of Protein Samples

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Cells were lysed in ice-cold RIPA cell lysis buffer (Millipore; 20-188) containing Complete Ultra Mini protease inhibitors (Roche; 05892970001). Lysates were vortexed, centrifuged at 13,000 rpm for 15 min, and homogenized using insulin syringes. Concentrations of proteins were estimated using Protein Assay Dye Reagent Concentrate (Biorad; 5000006). Samples containing equal amounts of protein were heated at 95°C for 5 min in LDS sample buffer (ThermoFisher; NP0007) containing 10% 2-Mercaptoethanol (Gibco; 31350-010) and separated by SDS-polyacrylamide gel electrophoresis (PAGE) using 4–12% NuPage Bis-Tris pre-cast gels (ThermoFisher; NP0322BOX) and MOPS buffer (ThermoFisher; NP0001). Proteins were transferred using transfer buffer (ThermoFisher; NP00061) and 0.45 μm PVDF membranes (Millipore; IPVH00010). Membranes were blocked for 30 min in 5% (w/v) non-fat milk and then sequentially incubated with primary and HRP-conjugated secondary antibodies. HRP activity was detected using SuperSignal West Pico Chemiluminescent Substrate (ThermoFisher; 34580).

Martin E.W., Chakraborty S., Presman D.M., Tomassoni Ardori F., Oh K.S., Kaileh M., Tessarollo L, & Sung M.H. (2019). Assaying Homodimers of NF-κB in Live Single Cells. Frontiers in Immunology, 10, 2609.

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Western Blot Analysis of Cell Lysates

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Cells were lysed in ice-cold RIPA cell lysis buffer (Millipore, 20–188) containing Complete Ultra Mini protease inhibitors (Roche; 05892970001). Lysates were vortexed, centrifuged at 18,000g for 15 min, and homogenized with insulin syringes. Sample proteins concentrations were equalized using the Protein Assay Dye reagent concentrate (Biorad; 5000006) and heated at 95ºC for 5 min in LDS sample buffer (ThermoFisher; NP0007) containing 10% 2-mercaptoethanol (Gibco; 31350–010). Proteins were separated by SDS-polyacrylamide gel electrophoresis (PAGE) using 4 to 12% NuPage Bis-Tris gels (ThermoFisher; NP0322BOX) and MOPS buffer (ThermoFisher; NP0001). Proteins were transferred using transfer buffer (ThermoFisher; NP00061) onto 0.45-μm PVDF membranes (Millipore; IPVH00010). Membranes were blocked in 5% (w/v) non-fat milk for 30 min and then sequentially incubated with primary and horseradish peroxidase (HRP)-conjugated secondary antibodies. HRP activity was detected using SuperSignal West Pico Chemiluminescent Substrate (ThermoFisher; 34580). Where indicated in the figure legends, membranes were stripped for 15 min with Restore Western Blot Stripping Buffer (21059).

Martin E.W., Pacholewska A., Patel H., Dashora H, & Sung M.H. (2020). Integrative analysis suggests cell type–specific decoding of NF-κB dynamics. Science signaling, 13(620), eaax7195.

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Quantitative Western Blot Analysis

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Protein concentrations were determined by BCA protein assay (Thermo Fisher Scientific, 23,225). Lysates (5 μg) were prepared in 4X LDS buffer (Invitrogen, NP0007) and 0.05 M DTT. Samples were boiled for 10 min at 95°C and separated by 1D-SDS-PAGE electrophoresis on a 12% Bis-Tris gel at 200 V for 60 min in MOPS running buffer and antioxidant (Invitrogen, NP0005). Transfer was performed on ice in a cold room at 350 mA constant for 1 h in transfer buffer (Invitrogen, NP00061) with 10% methanol onto a 0.2-μm PVDF membrane (Millipore, LC20002). After blocking in 5% milk in TBST, primary antibody (20 μg/ml; Novous, 100–2220) was incubated ON in 5% milk in TBST at 4°C. Membranes were incubated with secondary anti-rabbit (1:2500; GE Healthcare, NA934) or anti-murine (NXA931) HRP-coupled antibodies (1:2500; GE Healthcare) at RT for 2 h in 5% milk TBST solution. Protein bands were detected by chemiluminescence (Pierce ECL; Thermo Fisher Scientific, 32,106). ImageQuant TL (v2005, Amersham Biosciences) was used for densitometry analysis.

Bailus B.J., Scheeler S.M., Simons J., Sanchez M.A., Tshilenge K.T., Creus-Muncunill J., Naphade S., Lopez-Ramirez A., Zhang N., Lakshika Madushani K., Moroz S., Loureiro A., Schreiber K.H., Hausch F., Kennedy B.K., Ehrlich M.E, & Ellerby L.M. (2021). Modulating FKBP5/FKBP51 and autophagy lowers HTT (huntingtin) levels. Autophagy, 17(12), 4119-4140.

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