Cells were lysed in Laemmli sample buffer [2% SDS, 5% glycerol, 1.5% dithiothreitol (DTT), 0.01% Bromophenol Blue and 60 mM Tris-HCl pH 6.8]. Collected cells were sonicated (Diagenode) with three bursts of 15 s and heated for 10 min at 95°C. 10–15 μl of lysate was loaded on an SDS–polyacrylamide gel with a width of 1 mm, along with 7 μl of molecular weight markers (Bio-Rad). A voltage of 60 V for the stacking gel and 150 V for the resolving gel were applied. Gels were run in Tris-glycine electrophoresis buffer (25 mM Tris, 250 mM glycine and 0.1% SDS). For western blotting analysis, proteins were transferred to a 0.2 μm nitrocellulose membrane using the Trans-Blot Turbo Transfer System apparatus (Bio-Rad). The transfer was performed at 25 V for 7 or 10 min (according to the molecular weight of the proteins under investigation). Membranes were incubated with 5% skim milk in TBS-T buffer (TBS containing 0.1% Tween-20) for 1 h, followed by overnight incubation at 4°C with primary antibody and three washes with TBS-T before 1 h incubation at room temperature with the specific HRP-conjugated secondary antibody. Chemiluminescence detection was done by incubation with Luminata Classico or Crescendo (Millipore). Proteins were visualized by autoradiography on ECL films (Amersham) using various exposure times and manual development, or by using a Chemidoc imaging system (Bio-Rad).
Trans blot turbo transfer system apparatus
Manufactured by Bio-Rad
Sourced in United States
The Trans-Blot Turbo Transfer System is a laboratory equipment used for the rapid and efficient transfer of proteins from polyacrylamide gels to membranes. The system utilizes a combination of electrical current and pre-set transfer protocols to facilitate the transfer process, allowing for consistent and reproducible results.
Automatically generated - may contain errors
Lab products found in correlation
Luminata classico, by Merck Group (3 mentions)
Ecl film, by Cytiva (3 mentions)
Molecular weight marker, by Bio-Rad (3 mentions)
Trans blot turbo transfer pack, by Bio-Rad (2 mentions)
Chemidoc imaging system, by Bio-Rad (1 mentions)
Chemidoc xrs system, by Bio-Rad (1 mentions)
Trans blot turbo mini pvdf transfer pack, by Bio-Rad (1 mentions)
Mini protean tgx system precast protein gels, by Bio-Rad (1 mentions)
Erα sc 8002, by Santa Cruz Biotechnology (1 mentions)
5 protocols using trans blot turbo transfer system apparatus
1
SDS-PAGE and Western Blot Analysis
2
Western Blot Analysis of Protein Expression
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Cells were harvested and lysed in RIPA buffer. After 15 minutes on ice, samples were sonicated and centrifuged at 16,000 rpm for 10 minutes at 4°C. Protein concentration was measured using the BCA assay. 15ug of total proteins/sample was loaded in a precast 4–15% polyacrylamide gel (Bio-Rad #4561033EDU) and run at 100mV for 1 hour. Before transferring, gels were activated using UV to obtain total protein staining. Gels were then transferred on nitrocellulose filter paper (0.45μm pores; Bio-Rad #1620115) using a Trans-Blot Turbo Transfer System apparatus (Bio-Rad). Membranes were then imaged again for total protein and blocked in 5% milk solution in TBS-T 1X for 1 hour at room temperature. Membranes were then incubated o/n at 4°C with primary antibodies diluted in 5% BSA solution in TBS-T 1X. After two washes in TBS-T 1X at room temperature, membranes were incubated with a secondary antibody (1:10,000). Membranes were washed four times and then exposed to ECL solution (Thermo Fisher Scientific #34096). Images were acquired using ChemiDoc XRS+ System (Bio-Rad). The Image Lab software was used for WB quantification to quantify the band intensity for the protein of interest. Relative quantification was performed using total protein staining or loading control protein such as GAPDH/Actin. WB data were then processed to show a fold change compared to GFP.
3
SDS-PAGE and Western Blot Protocol
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Cells were lysed in Laemmli sample buffer [2% sodium dodecyl sulphate (SDS), 5% glycerol, 1.5% DTT, 0.01% bromophenol blue, 60 mM Tris HCl pH 6.8]. Collected cells were sonicated (Braun) with three bursts of 15 s and heated for 10 min at 95°C. A 6% SDS-polyacrylamide gel with a width of 1 mm was loaded with 15 μl of lysate, along with 6 µl of molecular weight markers (BioRad). Gels were run in Tris-glycine electrophoresis buffer (25 mM Tris, 250 mM glycine, 0.1% SDS) until the dye reached the bottom of the gel. For western blotting analysis proteins were transferred to a 0.2 µm nitrocellulose membrane (BioRad Trans-Blot Turbo transfer pack) using the Trans-Blot Turbo Transfer System apparatus (BioRad). The transfer was performed at 25 V for 10 min. Membranes were incubated with 5% skim milk in TBS-Tween buffer for 1 h, followed by overnight incubation at 4°C with primary antibody and three washes with TBS-Tween before 1 h incubation at room temperature with the specific HRP-conjugated secondary antibody. Chemiluminescence detection was done by incubation with Luminata Classico or Crescendo (Millipore). Proteins were visualized by autoradiography on ECL films (Amersham), using various exposure times and manually developed.
4
SDS-PAGE Immunoblotting for Protein Analysis
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Immunoblotting was performed ad described in Cabrini M. et al JCS 2021 [14] . Briefly, cells were lysed in Laemmli sample buffer (2% sodium dodecyl sulphate (SDS), 5% glycerol, 1.5% Dithiothreitol (DTT), 0.01% bromophenol blue, 60 mM Tris HCl pH 6.8). Protein solutions were sonicated (Diagenode) with three bursts of 15 sec and heated for 10 min at 95°C. A 6% SDS-polyacrylamide gel with a width of 1 mm was loaded with 15-20 μl of lysate, along with 7 µl of molecular weight markers (BioRad). Gels were run in Tris-Glycine electrophoresis buffer (25 mM Tris, 250 mM glycine, 0.1% SDS) until the dye reached the bottom of the gel. For western blotting analysis proteins were transferred to a 0.2 µm nitrocellulose membrane (BioRad Trans-Blot Turbo transfer pack) using the Trans-Blot Turbo Transfer System apparatus (BioRad). The transfer was performed at 25 V for 10 min. Membranes were incubated with 5% skim milk in TBS-Tween (0.1%) buffer for 1 h, followed by overnight incubation at 4°C with primary antibody and three washes with TBS-Tween before 1 h incubation at room temperature with the specific HRP-conjugated secondary antibody. Chemiluminescence detection was done by incubation with Luminata Classico or Crescendo (Millipore).
Proteins were visualized by autoradiography on ECL films (Amersham), using various exposure times and manually developed.
Proteins were visualized by autoradiography on ECL films (Amersham), using various exposure times and manually developed.
5
Western Blot Analysis of Apoptosis Markers
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After treating cells with test compounds for 24 or 48 h, cells were lysed in lysis buffer. Proteins were separated in 4-20% Mini-Protean TGX System Precast Protein Gels (Bio-Rad, Hercules, CA, USA) and transferred to Trans-Blot Turbo Mini PVDF Transfer Packs (Bio-Rad) using a Trans-Blot Turbo Transfer System apparatus (Bio-Rad). The blots were blocked for 1 h in 0.02 M Tris-buffered saline containing 5% bovine serum albumin and 0.1% Tween 20 and then incubated overnight at 4 °C with antibodies specific for caspase-3 and cleaved caspase-3 (#9662, Cell Signaling Technology), PARP (#9542, Cell Signaling Technology), ERα (sc-8002, Santa Cruz Biotechnology), ERβ (sc-8974, Santa Cruz Biotechnology), and GPR30 (ab3974, Abcam). The membranes were then washed three times in TBST (Tris-buffered saline, 0.1% Tween 20) and incubated for 1 h at room temperature with horseradish peroxidase (HRP)-conjugated antirabbit (#7074) secondary antibodies (Cell Signaling Technology). β-Actin (Cat. No. A5316, Sigma-Aldrich) was used as a loading control. Immunopositive bands were visualized using WesternBright Quantum HRP substrate (Cat. No. K-12043 D20, Advansta Inc., Menlo Park, CA, USA). Quantification of protein bands was performed by densitometry using VisionWorks LS Acquisition and Analysis software (UVP, LLC, Upland, CA, USA).
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