HEK293T cells were transfected with FLAG-PTBPs proteins. 48 hr post-transfection, cells were washed once with PBS and crosslinked with 150 mJ/cm2 UV light at 254 nm in Stratalinker 2400. Cells were collected by centrifugation and resuspended on lysis buffer (50 mM Tris–HCl pH 7.4, 100 mM NaCl, 1% IGEPAL CA-630, 0.1% SDS, 0.5% sodium deoxycholate, protease and phosphatase inhibitors). After sonication, lysates where digested with 4 U/ml Turbo DNase (Fisher, AM2238) and 1.5 U/μl RNase I (Fisher, 10330065). Lysates were centrifugated and supernatants incubated with pre-equilibrated α-FLAG M2 affinity gel beads for 2 hr at 4°C. Beads were washed twice with high-salt wash buffer (50 mM Tris–HCl, pH 7.4, 1 M NaCl, 1 mM EDTA, 1% IGEPAL CA-630, 0.1% SDS, 0.5% sodium deoxycholate, protease and phosphatase inhibitors). At that point, beads were equilibrated twice with PNK buffer (20 mM Tris–HCl, pH 7.4, 10 mM MgCl2, 0.2% Tween-20) and RNA 5′ ends where labeled with [γ-32P]-ATP and T4 PNK (NEB, M0201L) at 37°C for 5 min. Beads were eluded at 70°C for 5 min with NuPAGE loading buffer (Fisher, 11549166) and loaded in 4–12% NuPAGE Bis-Tris gel (Fisher, 10247002) for subsequent electrophoresis and transference to nitrocellulose membrane (Merck-Sigma, GE10600003). Precipitated RNA was analyzed by autoradiography and FLAG-PTBP protein levels were measured by western blot.
Nupage loading buffer
Manufactured by Thermo Fisher Scientific
Sourced in United States
NuPAGE loading buffer is a pre-formulated buffer used for preparing protein samples for electrophoresis analysis. It is designed to be compatible with the NuPAGE electrophoresis system.
Automatically generated - may contain errors
Lab products found in correlation
Ripa buffer, by Merck Group (5 mentions)
Nupage bis tris gel, by Thermo Fisher Scientific (5 mentions)
M per mammalian protein extraction reagent, by Thermo Fisher Scientific (5 mentions)
Pvdf pre cut blotting membranes, by Thermo Fisher Scientific (5 mentions)
Nupage novex 4 12 bis tris protein gel, by Thermo Fisher Scientific (4 mentions)
15 ml conical tube, by BD (4 mentions)
Transfer buffer, by Thermo Fisher Scientific (4 mentions)
Nupage sample reducing agent, by Thermo Fisher Scientific (4 mentions)
Mes buffer, by Thermo Fisher Scientific (4 mentions)
Western c ecl substrate, by Bio-Rad (4 mentions)
Horseradish peroxidase conjugated secondary antibody, by Merck Group (3 mentions)
Nupage 4 12 bis tris, by Thermo Fisher Scientific (3 mentions)
Hrp conjugated secondary antibody, by Agilent Technologies (3 mentions)
Protein g sepharose beads, by Merck Group (2 mentions)
Protease inhibitor, by Cell Signaling Technology (2 mentions)
Laemmli loading buffer, by Bio-Rad (2 mentions)
β mercaptoethanol, by Merck Group (2 mentions)
Ezview myc beads, by Merck Group (2 mentions)
Dynabeads protein g, by Thermo Fisher Scientific (2 mentions)
Complete protease inhibitor cocktail, by Roche (2 mentions)
48 protocols using nupage loading buffer
1
RNA-Protein Interaction Analysis via FLAG-PTBP Pulldown
2
Quantitative Protein Precipitation Analysis
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For qualitative analysis, 3 µl of the fraction analyzed was mixed with 47 µl water and precipitated in 150 µl cold acetone (Carl Roth GmbH & Co. KG; Karlsruhe, Germany) for at least 15 min. After centrifugation at 16,000 × g at 4°C for 10 min, the supernatant was removed, and the precipitate dried for 30 min at 45°C. The precipitate was resuspended in NuPage™ loading buffer (Fisher Scientific GmbH, Schwerte, Germany). Samples were incubated at 70°C for 10 min. Samples were loaded onto NuPage™ 10% Bis-Tris Gels (Fisher Scientific GmbH, Schwerte, Germany). The SeeBlue Pre-Stained marker (Fisher Scientific GmbH, Schwerte, Germany) was used as standard. Gels were run at 185 V for 35 min and then dried at 70°C (Unigeldryer 3545D, Uniequip, Planegg, Germany). The dry gels were placed on a phosphor screen (GE-Healthcare GmbH; Munich, Germany). After 1–2 days, the phosphor screen was scanned using an Amersham Typhoon RGB (GE-Healthcare GmbH; Munich, Germany) to visualize labeled proteins.
3
Protein Separation by SDS-PAGE
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Protein samples were prepared using NuPAGE loading buffer (Fisher Scientific UK Ltd., United Kingdom) and heated to 98°C for 10 min before loading the gel. Proteins were separated by SDS-PAGE using 4–12% precast Tris-Glycine SDS polyacrylamide gels (Fisher Scientific UK Ltd., United Kingdom) for 30 min at 200 V. SDS-PAGE loading used the equivalent of 50 μL of bacterial culture or 5 × 104 insect cells per lane of a 10 lane, 10 cm gel. After electrophoresis, gels were subjected to either Coomassie Brilliant Blue R250 staining or transferred to a polyvinylidene difluoride (PVDF) membrane for western blot analysis.
4
Protein Extraction and Co-Immunoprecipitation
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Tissue samples or cells were lysed in cold RIPA buffer supplemented with protease inhibitor (Cell signaling) and 1% Triton X-100 or 1% CHAPS in a tissue Precellys homogenizer (Bertin Corp.) (for tissue) or by trituration with a syringe (for cells). Protein concentration was assessed by bicinchoninic acid (BCA) assay (Novagen). For co-immunoprecipitation, protein extracts were incubated overnight at 4 °C with either Prdx2 antibody, EZview MYC beads (Sigma) or G protein sepharose beads (Sigma) only, as a control. Protein-antibody complexes were pulled down using G protein sepharose beads (Sigma) and washed with cold RIPA buffer. Immunoprecipitated proteins were incubated with Laemmli loading buffer (Bio-Rad) or NuPAGE loading buffer (Life Technologies) supplemented with 5% β-mercaptoethanol (Sigma) at 100 °C for 5 min.
5
Sortase-Mediated Amyloid-β Peptide Labeling
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A solution containing 70 µMCpSrtD was incubated with a 15 µM solution of a peptide comprising of the first 16 amino-acid residues of amyloid-β (Aβ1–16) fused at the C-terminus to a variety of sortase signal motifs in the presence of MES reaction buffer (50 mM MES pH 6.5, 200 mM NaCl, 1 mM TCEP) for 3 h at 316 K. The reaction was quenched by adding nonreducing NuPAGE loading buffer (Life Technologies). Following SDS–PAGE, resolved protein samples were transferred onto nitrocellulose membranes for Western blot analyses of thioacyl intermediate formation using an antibody against Aβ (WO2). Equal loading was determined by Western blot using anti-His5 antibody (Qiagen).
To analyze the impact of different metal ions on the catalytic activity of CpSrtD, recombinant protein was first incubated with 100 mM EDTA for 2 h at room temperature (RT). EDTA-treated CpSrtD was then diluted tenfold before being tested for catalytic activity in the presence of 10 mM metal ions for 3 h at 316 K. The reaction was quenched by the addition of nonreducing NuPAGE loading buffer. SDS–PAGE and subsequent Western blot analyses were then performed as above.
To analyze the impact of different metal ions on the catalytic activity of CpSrtD, recombinant protein was first incubated with 100 mM EDTA for 2 h at room temperature (RT). EDTA-treated CpSrtD was then diluted tenfold before being tested for catalytic activity in the presence of 10 mM metal ions for 3 h at 316 K. The reaction was quenched by the addition of nonreducing NuPAGE loading buffer. SDS–PAGE and subsequent Western blot analyses were then performed as above.
6
Western Blot Analysis of CEP83 and α-Tubulin
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Proteins were extracted from hiPSCs using radioimmunoprecipitation assay (RIPA) buffer (Sigma-Aldrich, R0278) as described in details in supplementary data. 30 µg protein in RIPA buffer were mixed with 1× reducing (10% b-mercaptoethanol) NuPAGE loading buffer (Life Technologies, Carlsbad, CA), loaded on a precast polyacrylamide NuPage 4–12% Bis-Tris protein gel (Invitrogen, Carlsbad, CA, USA) and blotted on 0.45 µm pore size Immobilon-P Polyvinylidene difluoride membrane (EMD Millipore, Billerica, MA; USA). The membrane was blocked in 5% bovine serum albumin for 1 hr at room temperature and incubated overnight at 4°C with primary antibodies: anti-CEP83 produced in rabbit (1:500, Sigma-Aldrich) and anti-α-Tubulin produced in mouse (1:500, Sigma-Aldrich, T9026). Then, the membrane was incubated for 1 hr at room temperature with horseradish peroxidase-conjugated secondary antibodies (1:2000, Sigma-Aldrich, Saint Louis, MO, USA). Chemiluminescent reagent (Super Signal–West Pico; Thermo Scientific, Waltham, MA; USA) was used to detect the proteins. The spectra Multicolor Broad Range Protein Ladder (Thermo Fisher Scientific, USA) was used to evaluate the molecular weight of corresponding protein bands.
7
Nef Protein Interaction Analysis
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Cells (2.8 × 105) were seeded on 60 mm dishes, cotransfected the following day with both 2.5 μg of either Nef-LAI or Nef-SF2 expressing plasmid and 4 μg of the ACOT8 constructs. Total DNA was compensated to the same amount with the pcDNA3 empty vector. Coimmunoprecipitation was performed 48 hours post-transfection as previously described60 (link). Briefly, cells were harvested and washed in ice cold PBS 1X, resuspended in 240 μl of non-denaturing lysis buffer (10 mM Tris–HCl, pH 7.5, 5 mM EDTA, 150 mM NaCl, 1% Triton X-100) and incubated for 10 minutes on ice. Lysates were sonicated twice for 5 seconds and frozen at −20 °C for 1 hour. Cellular debris was removed by centrifugation. Proteins obtained were quantified and incubated overnight at 4 °C with 2 μg of anti-HA antibody, and immunocomplexes were linked to Dynabeads® protein G (Life Technologies) at 4 °C for 30 minutes. Beads were washed with PBS 1X and resuspended in elution buffer containing NuPAGE loading buffer (Life Technologies) and 0.25 mM dithiothreitol for western blot analysis.
8
FLAG Affinity Purification Protocol
9
Protein Extraction and Co-Immunoprecipitation
10
Co-Immunoprecipitation of Protein Complexes
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HEK293T cells were harvested 24 h after transfection with 1 μg of each expression vector. Cells were lysed in non-denaturing buffer (10 mM Tris–HCl pH 7.5, 5 mM EDTA, 150 mM NaCl, 1% TritonX-100) supplemented with protease inhibitors Complete Protease Inhibitor Cocktail EDTA-free (Roche). Lysates were sonicated twice for 5 s, frozen at -80°C for 1 h and then centrifuged for 30 min at 14000 rpm at 4°C. Proteins were subjected to co-immunoprecipitation with the appropriate primary antibodies overnight at 4°C. Immunocomplexes were linked to magnetic beads of Dynabeads Protein G or A (LifeTechnologies) for 30 min at 4°C. The beads were then washed 3 times and resuspended in elution buffer containing NuPAGE loading buffer (LifeTechnologies) and 0.25 mM DTT.
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