Total protein was extracted from liver tissues using the RIPA buffer (sigma, R0278) and the Protease and phosphatase inhibitor cocktail (Thermo Scientific). After the concentration being quantified by BCA protein assay kit, equal amounts of protein from animals of the same group were pooled. Western blotting was performed as described previously27 (link). Rabbit anti-light chain 3 (LC3B; 1:1000, Abcam), rabbit anti-Caspase3 (1:1000, Cell signaling Technology), rabbit anti-SQSTM1/p62 (8025, 1:1000, Cell signaling Technology) and rabbit anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 1:10,000, Cell signaling Technology) were used as primary antibodies. Goat polyclonal antibody to rabbit IgG (1:5000) was used as secondary antibody. The membranes were probed using enhanced chemiluminescence western blotting substrate (GE Healthcare) and exposed to high sensitivity film (GE Healthcare) or digitalized with Fusion FX7 (Labtech International Ltd, Heathfield, United Kingdom). The signal intensity of each protein band was quantified using Gel Analyzer Module provided by ImageJ (National Institutes of Health, Bethesda, MD). All Western blots were repeated 3 times.
High sensitivity film
Manufactured by GE Healthcare
Sourced in United Kingdom
High sensitivity film is a laboratory imaging product designed to capture and record visual data with high resolution and clarity. It is a specialized film material optimized for enhanced sensitivity to light and other forms of radiation, enabling the capture of detailed images and data from a variety of laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Ripa buffer, by Merck Group (2 mentions)
Enhanced chemiluminescence western blotting substrate, by GE Healthcare (1 mentions)
Rabbit anti glyceraldehyde 3 phosphate dehydrogenase gapdh, by Cell Signaling Technology (1 mentions)
Protease and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Rabbit anti caspase 3, by Cell Signaling Technology (1 mentions)
Monoclonal anti β actin, by Merck Group (1 mentions)
Non fat milk, by Bio-Rad (1 mentions)
Acrylamide gel, by Bio-Rad (1 mentions)
Nitrocellulose membranes 0.45 μm, by Bio-Rad (1 mentions)
Polyvinylidene difluoride membrane, by GE Healthcare (1 mentions)
Anti nf κb p65, by Merck Group (1 mentions)
Rabbit anti caspase 3, by Merck Group (1 mentions)
Supersignal west femto trial kit, by Thermo Fisher Scientific (1 mentions)
Rabbit anti gapdh, by Merck Group (1 mentions)
Rabbit anti gapdh, by Cell Signaling Technology (1 mentions)
Ecl western blot substrate, by GE Healthcare (1 mentions)
Lc3b 2, by Novus Biologicals (1 mentions)
Bcl xl, by Abcam (1 mentions)
P s6 kinase, by Cell Signaling Technology (1 mentions)
P akt, by Cell Signaling Technology (1 mentions)
4 protocols using high sensitivity film
1
Evaluating Liver Protein Expression
2
Placental Protein Expression Analysis
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Placental tissues were obtained as villous, decidua, and membrane samples (first trimester), and as villous, basal plate, amnion, smooth chorion, and chorionic plate samples (second trimester and term). A placental diagram identifying these regions is available as Supplemental Figure S1 (Supplemental Data are available online at www.biolreprod.org ). Samples were homogenized using lysis buffer (150 mM NaCl, 50 mM Tris, 2 mM EDTA, 1% NP-40, 1× protease mix) with Western blotting carried out as previously described [20 (link)]. Briefly, samples were resolved by SDS-PAGE in 15% acrylamide gels (Bio-Rad) and transferred to nitrocellulose membranes (0.45 μm; Bio-Rad). Membranes were blocked in 3% nonfat milk (Bio-Rad) for 1 h followed by primary antibody incubation (anti-SDF2 [Sigma] polyclonal antibody was used at 2.5 ng/ml, polyclonal anti-GRP78 [Santa Cruz Biotechnology] at 0.15 μg/ml, polyclonal caspase 3 [Santa Cruz Biotechnology] at 0.6 μg/ml, and monoclonal anti-β-actin (clone AC-15; Sigma) were used as loading control at 0.15 μg/ml). Secondary antibodies (Jackson ImmunoResearch Laboratories) were used at 1.25 μg/ml. Immunoreactive bands were detected using the ECL method, and were captured with high-sensitivity film (GE Healthcare).
3
Protein Isolation and Immunoblotting Assay
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The total protein was isolated from liver tissues using RIPA buffer (Sigma-Aldrich, St. Louis, US). Ten micrograms of total protein was loaded per well and then separated on 12% sodium dodecyl sulfate-polyacrylamide gel. Then, the protein was transferred onto the polyvinylidene difluoride membrane (GE Healthcare, Buckinghamshire, United Kingdom). The membrane was blocked with 5% BSA in TBST (0.5% Tween 20) for 1 h at room temperature. The membrane was incubated at 4°C overnight with primary antibodies: rabbit anti-phospho-AKT (Ser473, 1 : 1000), rabbit anti-phospho-Stat3 (Tyr705, 1 : 2000), rabbit anti-phospho-NF-κB p65 (Ser536, 1 : 1000), rabbit anti-AKT (1 : 1000), rabbit anti-Stat3 (1 : 2000), anti-NF-κB p65 (1 : 1000), rabbit anti-caspase 3 (1 : 1000), and rabbit anti-GAPDH (1 : 20000, Sigma-Aldrich, St. Louis, USA). All antibodies besides rabbit anti-GAPDH were purchased from Cell Signaling Technology, Beverly, USA. The membrane was incubated for 1 h at room temperature with a goat polyclonal to rabbit IgG antibody (1 : 10000, Abcam) and developed with the SuperSignal West Femto Trial kit (Thermo, USA). The membrane was exposed to high-sensitivity films (GE Healthcare) for autoradiography.
4
Molecular Pathway Analysis by Western Blot
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Western blot analyses were performed as previously described [19] . After being blocked for nonspecific binding, membranes were incubated with primary antibodies against Rictor, Raptor, mTOR, P-S6 kinase (S6K), S6K (Cell Signaling Technology), Bcl-xl (Abcam), Bcl-2 (Abcam), Atg-5 (Abcam), LC3BII (Novus Bio), P-AKT (Cell Signaling Technology), AKT (Cell Signaling Technology), P-ERK (Cell Signaling Technology), ERK (Cell Signaling Technology), P-JNK (Cell Signaling Technology), GAPDH (Abcam), and β-actin (Sigma-Aldrich) at 4 °C with gentle shaking overnight. Then, the membranes were developed with ECL Western Blot Substrate (GE Healthcare) and exposed to high-sensitivity films (GE Healthcare) for autoradiography.
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