HCC cells (1 × 105) were grown in 6-well plates for 24 h. Cells were transfected with indicated plasmids or treated with anticancer drugs or autophagy inhibitors. Cells were washed with PBS and lysed with cell lysis buffer (Cell Signaling, Danvers, MA) containing protease inhibitor mixture (Roche, Indianapolis, IN) and PMSF (0.1 mM). Protein concentrations were determined using the Bio-Rad protein assay reagent (Bio-Rad, Hercules, CA). The proteins (40–60 µg) were separated by NuPAGE 4–12% Bis-Tris-SDS gel (Invitrogen) and transferred to a PVDF membrane (Thermo Scientific, Rockford, IL). Membranes were blocked in 1X blocking buffer (Sigma-Aldrich, St. Louis, MO) for 1 h and incubated with indicated primary antibodies for overnight at 4 °C. All antibodies were used as per the manufacturer’s suggestions. After washing the membranes three times with Tris-buffered saline with 0.1% Tween 20 (TBST), the membranes were incubated in the appropriate secondary antibody (1:10,000 dilution) (Jackson ImmunoResearch, PA) for 1 h at room temperature, and immunoreactive bands were visualized using ECL chemiluminescence detection reagents (Signagen Laboratories, Rockville, MD). The blots were developed appropriately with band detection within linear range, and intensities were quantified using ImageJ software (National Institutes of Health).
Nupage 4 12 bis tris sds gel
Manufactured by Thermo Fisher Scientific
NuPAGE 4%-12% Bis-Tris SDS gels are pre-cast polyacrylamide gels used for protein separation and analysis through SDS-PAGE. They feature a Bis-Tris buffer system and a 4-12% gradient of polyacrylamide for resolving a wide range of protein sizes.
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Lab products found in correlation
Nupage mops sds running buffer, by Thermo Fisher Scientific (3 mentions)
Protein assay reagent, by Bio-Rad (2 mentions)
Nupage mes sds running buffer, by Thermo Fisher Scientific (2 mentions)
Cell lysis buffer, by Cell Signaling Technology (1 mentions)
Blocking buffer, by Merck Group (1 mentions)
Pvdf membrane, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor mixture, by Roche (1 mentions)
Typhoon fla 9500 gel scanner, by GE Healthcare (1 mentions)
Enhanced chemiluminescent detection system, by Thermo Fisher Scientific (1 mentions)
Lysis buffer, by Cell Signaling Technology (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
N cadherin, by Cell Signaling Technology (1 mentions)
E cadherin, by Cell Signaling Technology (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Nupage 3 8 tris acetate protein gel, by Thermo Fisher Scientific (1 mentions)
6 protocols using nupage 4 12 bis tris sds gel
1
Western Blot Analysis of HCC Cells
2
PCNA-Ubiquitin Cleavage Kinetics Assay
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PCNA‐UbTAMRA cleavage assays were performed in a reaction buffer composed of 20 mM HEPES pH 7.5, 100 mM NaCl, 5 mM MgCl2, 0.25 mM ATP, 2 mM DTT. The cleavage reaction was started by addition of USP1 (±UAF1) followed by incubation at room temperature for the specified time course. The concentration of DUB used for the kinetic modelling experiments was 1, 0.5, 0.25 µM for USP1 and 20, 10, 5 nM for USP1‐UAF1. All the mutants tested for activity on PCNA‐UbTAMRA were in complex with UAF1, and they were run in parallel with wild‐type USP1 at a concentration of 25 nM. Samples were collected at the indicated time points, and the reaction was stopped by adding SDS loading buffer. Samples were loaded on NuPAGE 4–12% Bis‐Tris SDS gel (Invitrogen) and separated by running them at 180 V for 30 min. The TAMRA fluorescence signal was visualized using a Typhoon FLA‐9500 gel scanner (GE Healthcare), and the concentration of PCNA‐UbTAMRA and ubiquitin was quantified by comparing the TAMRA fluorescence of the individual bands with a calibration curve of TAMRA fluorescence in the same experimental setting.
3
Protein Separation by NuPAGE SDS-PAGE
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Proteins were separated on NuPAGE 4%–12% Bis-Tris SDS gels (Invitrogen) using NuPAGE MOPS SDS running buffer (Invitrogen) or NuPAGE MES SDS running buffer (Invitrogen) at 120 V for 1.5 h.
4
Western Blotting Analysis of miR-214 and PTK6 in Prostate Cancer
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Western blotting was performed as described previously71 (link). After transfection with miR-214 or NC mimic for 48 h, cell lysates were prepared from RWPE-2, PC3, DU145, MDA-PCa-2b, and LNCaP cells using lysis buffer (Cell Signaling Technology, Danvers, MA) containing a protease inhibitor cocktail (Roche, Indianapolis, IN). Cell lysates were also prepared from RWPE-2, PC3, DU145, and MDA-PCa-2b cells after transfection with empty vector or PTK6 plasmid for 24 h. Protein concentrations were determined using the Bio-Rad protein assay reagent (Bio-Rad, Hercules, CA). Cell lysates (40 µg) were separated on NuPAGE 4–12% Bis-Tris-SDS gels (Invitrogen) and then transferred to a polyvinylidene difluoride membrane (Millipore, Billerica, MA). The membrane was blocked in casein blocking buffer (1×) (Sigma-Aldrich, St. Louis, MO) and incubated with primary antibodies against cleaved PARP, PTK6, E-Cadherin, N-Cadherin, and GAPDH (Cell Signaling Technology) overnight at 4 °C. The following day, membranes were washed and subsequently incubated with the appropriate HRP-conjugated secondary antibodies for 1 h at room temperature. Following this incubation, membranes were washed and developed with an enhanced chemiluminescent detection system (Thermo Fisher Scientific, Waltham, MA).
5
SDS-PAGE and Native PAGE Protein Separation
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Protein samples were separated by electrophoresis on NuPAGE 4%-12% Bis-Tris SDS gels (Invitrogen) using NuPAGE MOPS SDS running buffer (Invitrogen) at 200 V.
Native PAGE Protein samples were separated by electrophoresis on NuPAGE 3%-8% Tris-acetate protein gels (Invitrogen) using NPage buffer (50 mM Tris and 38 mM glycine) at 150 V at 4 C.
Native PAGE Protein samples were separated by electrophoresis on NuPAGE 3%-8% Tris-acetate protein gels (Invitrogen) using NPage buffer (50 mM Tris and 38 mM glycine) at 150 V at 4 C.
6
SDS-PAGE Protein Separation Optimization
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Protein samples were separated by electrophoresis on SDS polyacrylamide gels consisting of running gels (380 mM Tris-HCl pH 8.8, 0.1% SDS, 8%-15% of acrylamide, 0.21 À0.40% bis-acrylamide) and stacking gels (60 mM Tris-HCl pH 6.8, 0.1% SDS, 5% acrylamide, 0.13% bis-acrylamide). Proteins were separated with SDS-PAGE running buffer (25 mM Tris, 250 mM glycine, 0.1% SDS) at 40 mA. For analyses of NS-proteins and engineered CAT-tailed proteins (except for Figures S4H andS5D), proteins were separated on NuPAGE 4%-12% Bis-Tris SDS gels (Invitrogen) using NuPAGE MOPS SDS running buffer (Invitrogen) or NuPAGE MES SDS running buffer (Invitrogen) at 200 V.
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