RIPA lysis buffer (Solarbio, China) containing protease inhibitor cocktail (AbMole, USA) and phosphatase inhibitor cocktail (AbMole, USA) was added to 30 mg of tissue, and then the tissue was ground (40 m/s, 20 s, twice) using a homogenizer (MP Biomedicals, USA). The homogenate was incubated at 4 °C for 30 min and then centrifuged at 16,000 g at 4 °C for 10 min. The supernatant was the tissue protein sample. The protein concentration of the tissue protein sample was determined using a BCA Protein Assay Kit (Solarbio, China), and the protein concentration was adjusted to 4 mg/mL by adding loading buffer (NCM Biotech, China). The protein sample was denatured by incubation at 100 °C for 10 min, and WB was performed to detect the expression of proteins in tumor tissue. Information on the antibodies used in WB experiments is shown in Additional file 1 : Table S4.
Protease inhibitor cocktail
Manufactured by AbMole
Sourced in United States
Protease inhibitor cocktail is a laboratory product designed to inhibit the activity of various proteases. It is a mixture of chemical compounds that target and inactivate different types of proteases, preventing them from breaking down proteins. The core function of this product is to preserve the integrity of protein samples during experimental procedures.
Automatically generated - may contain errors
Lab products found in correlation
Phosphatase inhibitor cocktail, by AbMole (2 mentions)
Bca protein assay kit, by Solarbio (1 mentions)
Ripa lysis buffer, by Solarbio (1 mentions)
Anti ripk1, by Cell Signaling Technology (1 mentions)
0.45 μm nitrocellulose membrane, by Merck Group (1 mentions)
Anti p mlkl, by Abcam (1 mentions)
Ecl kit, by Merck Group (1 mentions)
Ripa buffer, by Solarbio (1 mentions)
Anti gapdh, by Abcam (1 mentions)
Anti ripk3, by ProSci (1 mentions)
Vegfa, by ABclonal (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Bca kit, by Meilun (1 mentions)
Hif 1α, by ABclonal (1 mentions)
β actin, by ABclonal (1 mentions)
Parp1, by Abcam (1 mentions)
P iκbα, by Abcam (1 mentions)
Pvdf membrane, by GE Healthcare (1 mentions)
Hrp conjugated secondary antibody, by Beyotime (1 mentions)
Pvdf membrane, by Beyotime (1 mentions)
5 protocols using protease inhibitor cocktail
1
Protein Extraction and Western Blot Analysis
2
Western Blot Analysis of RIPK1, RIPK3, and p-MLKL
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Lysates of heart tissue and cultured cells were prepared in RIPA buffer (Solarbio) supplemented with a protease inhibitor cocktail (Abmole, Houston, USA). The total protein content (80 μg) was segregated on 12% SDS–polyacrylamide gels and was then transferred onto 0.45 μm nitrocellulose membranes (Millipore, Plano, TX, USA). After being blocked in PBS-T buffer containing 5% fat-free milk at 37 °C for 2 h, the membranes were incubated with rabbit anti-RIPK1 (1:1200, Cell Signaling, Danvers, USA), anti-RIPK3 (1:2000, ProSci, San Diego, USA), anti-p-MLKL (1:2000, Abcam, Cambridge, MA, USA) or anti-GAPDH (1:2000, Abcam) antibodies at 4 °C overnight. After incubation with the horseradish peroxidase-conjugated secondary antibodies, the protein bands were visualized using an ECL kit (Sigma, USA).
3
Western Blot Analysis of Liver Markers
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Cells were dissolved in a RIPA lysis buffer, supplemented with protease inhibitor cocktail (Abmole), and after centrifugation at 12,000 g for 10 min at 4 °C, the supernatant was collected. The proteins were then resolved by 10% SDS-PAGE gel and electro-transferred to nitrocellulose membranes (EMS Millipore). The membrane was blocked with 5% skim fat milk in PBS with 0.1% Tween 20 for 1 h at room temperature, and incubated with the relevant primary antibodies overnight at 4 °C. Following washing in TBS-T, the membranes were incubated with the appropriate secondary antibody for 1 h at room temperature. Human GAPDH was used as the control of protein loading. Antibody used for western blot were as follows: rabbit anti-human AFP (1:1,000), rabbit anti-human ALB (1:5,000), mouse anti-human A1AT (1:100), rabbit anti-human HNF4α (1:1,000), mouse anti-human NTCP (1:1,000), mouse anti-human FXR (1:1,000), goat anti-rabbit IgG-HRP secondary antibody (1:10,000), goat anti-mouse IgG-HRP secondary antibody (1:10,000), monoclonal antibodies against human GAPDH were used as a control of protein loading. Detailed information of all antibodies is given in Table 2 .
4
Protein Extraction and Western Blot Analysis
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Total protein was extracted from theca layer and granulosa layer using RIPA Lysis Buffer (Beyotime, Shanghai, China; P0013B) and Protease Inhibitor Cocktail (AbMole, Shanghai, China; M5293), Phosphatase Inhibitor Cocktail (AbMole, Shanghai, China; M7528). Total protein content was determined using a BCA kit (Meilunbio, Dalian, China; MA0082). Proteins were transferred to Polyvinylidene fluoride membranes following migration through SDS-PAGE gels of gradient concentrations. Following blocking with skimmed milk powder, membranes were incubated with primary antibodies to protein to detect CD31 (1:1000, ABclonal, Wuhan, China; A0378), VEGFA (1:200, ABclonal, Wuhan, China; A12303), VEGFR (1:500, ABclonal, Wuhan, China; A11127), P-VEGFR (1:500, ABclonal, Wuhan, China; AP0382), HIF1α (1:1000, ABclonal, Wuhan, China; A0378), β-actin (1:2000, ABclonal, Wuhan, China; AC038). Secondary antibodies were combined with primary antibodies and protein bands were visualized with a chemiluminescence system (Tanon, Shanghai, China) and quantified with Image J software.
5
Protein Expression Analysis by Western Blot
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Cells were washed and lysed using cell lysis buffer RIPA (Beyotime) supplemented with 1 mM Protease Inhibitor Cocktail (AbMole) and 1 mM Phosphatase Inhibitor Cocktail (APExBIO, Houston, USA). Cell lysates were subject to SDS/polyacrylamide gel electrophoresis and transferred to a PVDF membrane (GE Healthcare Life sciences, Braunschweig, Germany) using a semi-dry transfer apparatus (Bio-Rad, Hercules, USA). The membrane was blocked with 5% bovine serum albumin (BSA) for 1 h at room temperature, and subsequently incubated with specific antibodies against p-ATM, p-IκBα, XIAP, NEMO, H2AX, p65 or PARP-1 (Abcam) diluted in antibody dilution buffer at 4°C overnight. PVDF membranes were washed with PBS supplemented with 0.05% Tween-20 (PBST) and incubated with HRP-conjugated secondary antibodies (1:5000; Beyotime) for 1 h at room temperature. Finally, signals were visualized using Chemiluminescence Imaging System ChemiScope6000 (Clinx, Shanghai, China). GAPDH was used as a loading control. Intensity was quantified by Image J software.
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