Total proteins (30 μg) were separated by SDS/polyacrylamide gel electrophoresis (10% acrylamide gel) using the Bio-Rad Mini-Protein II System. Proteins were transferred to expanded polyvinylidene difluoride membranes (Pierce, Rockford, IL, USA). Following blocking, the membrane was probed with the primary antibodies. The blots were incubated with goat polyclonal antibody (1 : 1000) to bind actin, which served as the internal control. After removal of the primary antibody, the blots were incubated for 2 h at room temperature with the appropriate peroxidase-conjugated secondary antibody and then developed by autoradiography using an ECL-Western blotting system (Amersham International, Buckinghamshire, UK). The immunoblots for MLC2 (19 kDa) and phospho-MLC-2 (19 kDa) were quantified with a laser densitometer.
Mini protein 2 system
Manufactured by Bio-Rad
Sourced in United States
The Mini-Protein II System is a compact, vertical electrophoresis system designed for separating protein samples. It features a simple design and can accommodate a variety of gel formats. The core function of this system is to facilitate the separation and analysis of protein samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti pepck, by Santa Cruz Biotechnology (1 mentions)
Anti β actin, by Merck Group (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Trans blot system, by Bio-Rad (1 mentions)
Protein assay, by Bio-Rad (1 mentions)
Image labtm software version 5, by Bio-Rad (1 mentions)
Chemidoc imaging system, by Bio-Rad (1 mentions)
β actin, by Thermo Fisher Scientific (1 mentions)
Trans blot turbo system, by Bio-Rad (1 mentions)
Dithiothreitol (dtt), by Cell Signaling Technology (1 mentions)
Enhanced chemiluminescent solution, by Bio-Rad (1 mentions)
Phospho akt, by Cell Signaling Technology (1 mentions)
Phospho s6 ribosomal protein, by Cell Signaling Technology (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
6 protocols using mini protein 2 system
1
Western Blot Analysis of Contractile Proteins
2
Immunoblotting of PEPCK and TGR5 Proteins
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To determine the effect of antidiabetic drugs on glucose and lipid metabolism in diabetic models, the protein expression levels of PEPCK and TGR5 were measured [36 (link)]. Total proteins (30 µg) prepared from tissue homogenates were separated through SDS/polyacrylamide gel electrophoresis (10% acrylamide gel) using the Bio-Rad Mini-Protein II System. Proteins were transferred to expanded polyvinylidene difluoride membranes (Millipore, Burlington, MA, USA). Following blocking, the membrane was probed with the primary antibodies. After removal of the primary antibody, the blots were incubated for 1 h at room temperature with the appropriate peroxidase-conjugated secondary antibody and then developed using the ECL-Western Blotting System (Amersham International, Buckinghamshire, UK). Antibodies used for immunoblotting were anti-PEPCK (62 kDa, Santa Cruz Biotechnology, Dallas, Tx, USA) and anti-TGR5 receptor protein (32 kDa, Abcam, Cambridge, MA, USA). Anti-β-actin (43 kDa, Sigma-Aldrich, St. Louis, MO, USA) was used as a loading control.
3
Protein Extraction and Western Blot Analysis
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We used the ice-cold radioimmunoprecipitation assay (RIPA) buffer to extract the protein from tissue homogenates or cell lysates as described in our previous method [16 (link)]. The protein level was characterized using Biorad protein assay (Biorad Laboratories, Inc., Hercules, CA, USA). After separation of proteins (30 μg) by SDS/polyacrylamide gel electrophoresis (10% acrylamide gel) through a Biorad Miniprotein II system, it was transferred to the expanded polyvinylidene difluoride membranes (Pierce, Rockford, IL, USA) with a Biorad Trans-Blot system. Then, the membranes were washed and blocked for 1 h at room temperature with 5% (w/v) skimmed milk powder according to our previous method [16 (link)]. The primary antibody reactions were performed following the manufacturer's instructions. The blots were incubated with goat polyclonal antibody (1 : 1000) to bind actin that served as the internal control. After removal of primary antibody, the washed blots were incubated with the appropriate peroxidase-conjugated secondary antibody for 2 h at room temperature. The blots were then developed using an ECL-Western blotting system (Amersham International, Buckinghamshire, UK). Each immunoblot, including PPARδ (50 kDa), cardiac troponin I (28 kDa), or phospho-troponin I (28 kDa), was then quantified by a laser densitometer.
4
Western Blot Analysis of Striatal Protein Signaling
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After protein extraction from striatal tissues using RIBA lysis buffer (Bio Basic Inc., Markham Ontario, L3R 8T4 Canada), equal amounts of protein were separated on a poly-acryl amide gel by SDS-PAGE using a Bio-Rad Mini-Protein II system. Polyvinylidene difluoride membranes was used to transfer the protein (Pierce, Rockford, IL, USA) with a Bio-Rad Trans-Blot Turbo system. Immunodetection of Western blots was conducted by incubating the membranes at room temperature for 1 h with blocking solution composed of 20 mM Tris–Cl (pH 7.5), 150 mM NaCl, 0.1% Tween 20 and 3% bovine serum albumin. Membranes were incubated overnight at 4 °C with one of the following primary antibodies (1:1000): p-(Tyr 1022/1023) JAK1 (Cat. no. 700028), p-(Tyr 1007–1008) JAK2 (Cat. no. PA5-85,735), p-(Tyr 705) STAT3 (Cat. no. 710093) and β-actin (Cat. no. MA5-15,739) obtained from Thermo Fisher Scientific Inc. (Rockford, IL). After washing, peroxidase-labeled secondary antibodies were added, and the membranes were incubated at 37 °C for 1 h. Analysis of the band intensity was performed using ChemiDoc™ imaging system with Image LabTM software version 5.1 (Bio-Rad Laboratories Inc., Hercules, CA, USA). The results are expressed as arbitrary units after normalization to β-actin protein expression.
5
Western Blot Analysis of Signaling Proteins
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Cells were washed with PBS and lysed in blue loading buffer containing dithiothreitol (Cell Signaling Technology) following the manufacturer's protocol. Proteins in cell lysates were separated on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis gels and transferred onto polyvinylidene difluoride membranes using a Mini-Protein II system (Bio-Rad). After incubating for 2 h with 5% non-fat milk blocking buffer, membranes were incubated at 4 °C overnight with rabbit primary antibodies for AKT, S6 ribosomal protein, 4E-BP1, MEK1/2, ERK1/2, phospho-AKT (Ser473), phospho-S6 ribosomal protein (Ser235/236), phospho-4E-BP1 (Thr70), phospho-MEK1/2 (Ser217/221), phospho-ERK1/2 (Thr202/Tyr204), phospho-p90RSK (Ser380), or β-actin (Cell Signaling Technology). Membranes were incubated for 2 h with horseradish peroxidase-linked goat anti-rabbit antibody. Protein bands were detected with enhanced chemiluminescent solution (Bio-Rad) on a GeneGnome HR image capture (Cambridge). Quantitative analysis of protein bands was carried out using GeneTools software.
6
Western Blot Immunodetection of Canine Antibodies
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The whole-cell protein preparation (200 μg) was separated by 1-dimensional 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; 1-DE), as described by Laemmli [21 (link)], using the Mini-Protein II System (Bio-Rad, Hercules, CA, USA) at 200 V. Proteins were electroblotted onto nitrocellulose membranes. Blotted membranes were blocked with 5% non-fat dried milk in TBS-T for 1 h at room temperature and were then incubated overnight at 4°C with serum samples from each dog that were diluted 1:500 or 1:1,000. Membranes were washed 3 times with TBS-T and incubated with rabbit peroxidase-conjugated anti-dog IgG (Cat. No. A9042, Sigma, St. Louis, MO, USA), IgG1 (Cat. No. AHP947P, AbD Serotec, Kidlington, UK) or IgG2 (Cat. No. AHP948P, AbD Serotec) at a dilution of 1:10,000 for 1 h. Reactive bands were detected using Metal Enhanced DAB reagent (Thermo Scientific, Rockford, IL, USA). After optimization experiments based on reactivity and specificity, asymptomatic dog serum samples were used at a final dilution of 1:500 and symptomatic dog serum samples were used at a final dilution of 1:1,000 (S1 Fig ).
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