Analysis was performed with whole cell extracts as previously described (5 (link)). Briefly, cells were lysed in 4× Laemmli Buffer (20% glycerol, 4% SDS, 100 mM Tris (pH 6.8), fresh 1 mM DTT) and boiled at 100°C for 10 min. Aliquots were separated on pre-cast 4–20% Tris-glycine gels (Bio-Rad Mini-PROTEAN TGX System) at 20 mA constant current. Protein was transferred to Immobilon-P (Millipore) with a Bio-Rad semi-dry transfer apparatus. Membranes were blocked for 45 min using 10% evaporated milk (Carnation) and 90% phosphate-buffered saline with 0.1% Tween (PBST). The membranes were incubated with primary antibodies MLH1 (BD Transduction Labs), PMS2 (BD Transduction Labs) and beta-actin (Sigma) in 10% milk/90% PBST for 90 min at room temperature or overnight at 4°C. After several washes with PBST the membranes were incubated for 45 min with horseradish peroxidase-conjugated goat anti-mouse (or goat anti-rabbit) secondary antibody (Pierce) in 10% milk / 90% PBST. After several PBST washes, the ECL Advance chemiluminescence kit (Amersham) was used to produce a signal captured by a Kodak Gel Logic 440 Imaging System. Image analysis was done with Carestream Molecular Imaging Software version 5.0.2.26 for Mac OS. Statistical analysis was done using the Student's t-test for unpaired data with unequal variance.
Mini protean tgx system
Manufactured by Bio-Rad
Sourced in United States
The Mini-PROTEAN TGX System is a pre-cast polyacrylamide gel electrophoresis (PAGE) system designed for rapid protein separation and analysis. The system includes pre-cast gels, running buffers, and accessories necessary for performing PAGE experiments. The system is capable of separating proteins based on their molecular weights.
Automatically generated - may contain errors
Lab products found in correlation
Immobilon p, by Merck Group (1 mentions)
Gel logic 440 imaging system, by Kodak (1 mentions)
β actin, by Merck Group (1 mentions)
Wide range molecular weight markers, by Merck Group (1 mentions)
Bio safe coomassie stain, by Bio-Rad (1 mentions)
Glass bead, by Merck Group (1 mentions)
Precellys 24 bead beater, by Bertin Technologies (1 mentions)
Anti strep tag 2 antibody, by Abcam (1 mentions)
Page blue protein stain, by Thermo Fisher Scientific (1 mentions)
Proteasearrest protease inhibitor cocktail, by G Biosciences (1 mentions)
Trans blot turbo transfer pack and system, by Bio-Rad (1 mentions)
Dc protein assay, by Bio-Rad (1 mentions)
Mini protean tgx stain free gel, by Bio-Rad (1 mentions)
Laemmli buffer, by Bio-Rad (1 mentions)
Tgx 4 15, by Bio-Rad (1 mentions)
Rabbit monoclonal, by Abcam (1 mentions)
Restore western blotting stripping buffer, by Thermo Fisher Scientific (1 mentions)
Brachidonic acid assay kit, by Thermo Fisher Scientific (1 mentions)
Rabbit igg, by Cell Signaling Technology (1 mentions)
Ne per nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (1 mentions)
5 protocols using mini protean tgx system
1
Immunoblotting Analysis of Mismatch Repair Proteins
2
Protein Electrophoresis Protocol
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Total protein and the albumin and globulin fractions were electrophoretically separated in 15% polyacrylamide gels under denaturing and reducing conditions (SDS-PAGE). Wide Range Molecular Weight Markers (Sigma-Aldrich, Cat. S8445, 6500−200,000 Da) were used, and 20 µg of test protein were loaded per lane. The electrophoresis was run in a Mini-PROTEAN®TGX system (Bio-Rad, Hercules, CA, USA) at an initial voltage of 70 V during the first 10 min and at 200 V for additional 2 h. A commercial running buffer was used (Bio-Rad, Cat. 1610732). The gels were stained using Bio-Safe Coomassie stain (Bio-Rad, Cat. 161-0786), and the resulting gel image was digitalized.
3
Synechocystis Protein Extraction and Analysis
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To extract proteins from Synechocystis, 2 ml of culture was centrifuged at 5000 x g for 10 min at 4 °C. The pellet was washed once and then resuspended with 200 μl of buffer containing 50 mM Hepes-NaOH, pH 7.5, 30 mM CaCl2, 800 mM sorbitol and Protease Arrest™ Protease Inhibitor Cocktail (100x, G-Biosciences). The cells were disrupted by acid-washed glass beads (425–600 μm diameter, Sigma-Aldrich) using the Precellys-24 Beadbeater (Bertin Instruments), program 3 × 30 s. Protein concentration was determined with the DC protein assay (Bio-Rad). 10 μg of total protein was applied to SDS-PAGE, if nothing else is stated.
Proteins in crude extracts were separated according to their molecular size with Mini Protean TGX Stain free gels (any kDa, Bio-Rad). The gels were run using a Mini-PROTEAN TGX™ system (Bio-Rad) for 1.5 h at 150 V. For visualizing all proteins and to control equal loading, the gels were stained using Page Blue Protein stain (Thermo Scientific). For detecting Strep-tagged proteins, the SDS-gels were blotted to PVDF membranes using the Trans-Blot turbo transfer pack and system (Bio-Rad) according to manufacturer’s instructions. Membranes were blocked in 5% milk for 2 h. Detection of the proteins was done using an anti-Strep-tag II antibody (Abcam, ab76949).
Proteins in crude extracts were separated according to their molecular size with Mini Protean TGX Stain free gels (any kDa, Bio-Rad). The gels were run using a Mini-PROTEAN TGX™ system (Bio-Rad) for 1.5 h at 150 V. For visualizing all proteins and to control equal loading, the gels were stained using Page Blue Protein stain (Thermo Scientific). For detecting Strep-tagged proteins, the SDS-gels were blotted to PVDF membranes using the Trans-Blot turbo transfer pack and system (Bio-Rad) according to manufacturer’s instructions. Membranes were blocked in 5% milk for 2 h. Detection of the proteins was done using an anti-Strep-tag II antibody (Abcam, ab76949).
4
Caveolae Proteomic Analysis Workflow
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Caveolae were isolated and prepared for the mass spectrometry analysis in three independent experiments. A volume of 15 μl from the selected gradient aliquot was mixed with an equal volume of Laemmli buffer (BIO-RAD Laboratories, USA) and then loaded onto SDS precast gel TGX 4–15% (BIO-RAD Laboratories). Proteins were separated on the gel using a Mini-protean TGX system (BIO-RAD Laboratories, USA), bathed in running buffer solution (tris/glycine/SDS 1X buffer BIO-RAD Laboratories, USA). Gels were then incubated overnight in Coomassie blue for protein staining and fixation and washed in ultrapure water allowing several changes. The gel lanes were excised and separated into three fragments: roughly above 75 kDa, below 25 kDa and between 25 and 75 kDa. The fragments were then submitted to mass spectrometer. For the purpose of the analysis, the three bioinformatic files representing the fragments were subsequently reunited for the data analysis. When a protein was detected in more than one fragment, the peptide with the maximum counts was retained.
5
Alkbh1 Protein Expression Analysis
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Western blotting was performed as previously described 32 . Briefly, cells were collected and lysed in NE-PER nuclear and cytoplasmic extraction reagent (Thermo Fischer, Cat# 78833). The protein content was determined using the Brachidonic-acid assay kit (Thermo Fisher Scientific, Cat# 23227). Equal loads of proteins were separated with the Mini-PROTEAN TGX system (Bio-Rad Laboratories) and transferred to polyvinylidene difluoride membrane (Bio-Rad Laboratories). Membranes were incubated with antibody against Alkbh1 (1:500, Rabbit monoclonal, abcam, Cat# ab195376) overnight at 4 . After incubation with horseradish peroxidase-conjugated secondary antibody (IgG detector, Takara Clontech, Cat# T7122A-1), the antigen was detected using chemiluminescence Western blotting detection reagents (Pierce ECL Western Blotting Substrate, Thermo Fisher Scientific Inc., Rockford, IL, the USA, Cat# 32106).
The image was scanned with ChemiDoc (Bio-Rad Laboratories). Membranes were striped using stripping buffer (Restore Western Blotting Stripping Buffer, Thermo Fisher Scientific Inc., Cat# 21059) and re-probed using anti-beta actin (1:5000, Rabbit IgG, Cell Signaling Technology, Cat# 13E5) using the same steps that were used previously. Semi-quantification was performed utilizing the ImageJ software after correcting the signal to an internal control (beta-actin).
The image was scanned with ChemiDoc (Bio-Rad Laboratories). Membranes were striped using stripping buffer (Restore Western Blotting Stripping Buffer, Thermo Fisher Scientific Inc., Cat# 21059) and re-probed using anti-beta actin (1:5000, Rabbit IgG, Cell Signaling Technology, Cat# 13E5) using the same steps that were used previously. Semi-quantification was performed utilizing the ImageJ software after correcting the signal to an internal control (beta-actin).
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