Protein samples were homogenized in a lysis buffer (Beyotime, Suzhou, China) containing proteinase inhibitors and phosphatase inhibitors (Selleck, TX, USA). According to the manufacturer’s protocol, the protein concentration was evaluated using a protein assay reagent kit (Beyotime). Equal amounts of proteins were separated on a sodium dodecyl sulfate polyacrylamide gel and transferred onto polyvinylidene fluoride membranes (Merck Millipore, MA, USA). The membranes were then incubated with the primary antibodies anti-DHCR24 (1:500) (Thermo Fisher Scientific, MA, USA), anti-STAT3 (1: 1,000, Proteintech, IL, USA), anti-β-actin (1: 3,000, Sigma). The proteins were detected using ECL Western Blotting Detection Reagents (Millipore) in accordance with the supplier’s protocol.
Proteinase inhibitor
Manufactured by Selleck Chemicals
Sourced in United States, China
Proteinase inhibitor is a type of laboratory equipment used to prevent the activity of proteolytic enzymes, known as proteases, which can degrade proteins. It is commonly used in protein purification and analysis procedures to preserve the integrity of the target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Phosphatase inhibitor, by Selleck Chemicals (2 mentions)
Polyvinylidene fluoride (pvdf), by Merck Group (2 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions)
Protein assay reagent kit, by Beyotime (1 mentions)
Ecl western blotting detection reagent, by Merck Group (1 mentions)
Anti stat3, by Proteintech (1 mentions)
Anti dhcr24, by Thermo Fisher Scientific (1 mentions)
Anti β actin, by Merck Group (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Lysis buffer, by Beyotime (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Radioimmunoprecipitation assay buffer, by Beyotime (1 mentions)
Enhanced chemiluminescence kit, by Merck Group (1 mentions)
Polyvinylidene di uoride membrane, by Merck Group (1 mentions)
Ecl kit, by Merck Group (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Ab168377, by Abcam (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Bca assay kit, by Beyotime (1 mentions)
7 protocols using proteinase inhibitor
1
Analyzing Protein Expression Using Western Blotting
2
Western Blot Analysis of ARPE-19 Cells
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ARPE-19 cells were lysed in radioimmunoprecipitation assay buffer (Beyotime, Shanghai, China) supplemented with proteinase inhibitors and phosphatase inhibitors (Selleck, Houston, TX, USA). Equal amounts of protein (30 μg) were loaded and separated on 10% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (EMD Millipore, Billerica, MA, USA). The membranes were blocked with 5% nonfat milk and were immunoblotted with primary antibodies at 4°C overnight. After washing in PBS-T (PBS containing 0.1% Tween-20) three times, the membranes were incubated with horseradish peroxidase (HRP)-labeled secondary anti-rabbit or anti-mouse antibodies for 1 h at room temperature. Proteins of interest were visualized using an enhanced chemiluminescence kit (EMD Millipore), and band intensities were quantified by densitometry using ImageJ software (National Institutes of Health, Bethesda, MD, USA). More detailed information on the antibodies used for western blot assays is listed in Supplementary Table 2 .
3
Western Blot Protein Detection Protocol
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Cells were lysed with RIPA (Radio Immunoprecipitation Assay) lysis buffer, including proteinase inhibitors (Bimake, Houston, TX, USA, NO. B14002). A BCA assay was performed to determine the concentrations of protein. Proteins were separated by electrophoresis on SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) gels (8%, 10%, or 15%), shifted to PVDF (polyvinylidene fluoride) membranes, and the membranes were incubated for 2 h with 5% skimmed milk at room temperature. After the membranes were incubated with the primary antibodies overnight at 4 °C, the corresponding HRP (horseradish peroxidase)-conjugated secondary antibody was added and incubated for another 2 h. The signals were obtained subsequent to the reaction with the HRP substrate. The information for all antibodies is shown in Table 1.
4
Western Blot Analysis of Cellular Proteins
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PC12 cells from each group were washed with ice-cold PBS, and total protein was extracted with RIPA lysis buffer (Beyotime Biotechnology Co., China) containing 1% proteinase inhibitors (Bimake, USA). The protein concentrations were measured using a BCA protein assay kit (Beyotime Biotechnology Co., China). Equal amounts of protein samples were subjected to separation by 10% or 12% SDS-PAGE and transferred to a poly-vinylidene di uoride membrane (Millipore). Next, 5% nonfat slim milk was used to block the membranes. After washing three times with TBST, the primary antibodies against ACSL4, GPX4, PEBP1, GAPDH (1: 1 000; A nity) were used to perform WB analysis. The membranes were then incubated with the corresponding primary antibodies overnight at 4°C. After the membranes were washed three times with TBST, they were incubated for 1 h at room temperature with goat anti-rabbit IgG polyclonal secondary antibody (1:5,000-1:10,000). Last, the signals were measured with an enhanced chemiluminescence (ECL) kit (Millipore, USA) on a gel imaging system.
5
Quantitative Western Blot Analysis
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Cells were collected and washed twice with PBS; the Radioimmunoprecipitation assay buffer (RIPA buffer) in the presence of proteinase inhibitor (Selleck Chemicals, Houston, TX, USA) was added. The crude lysates were transferred to pre-chilled Eppendorf tubes and centrifuged at 12,000 × g for 15 min at 4°C. The whole-cell lysates were resolved on a 10% SDS-polyacrylamide gel and electrophoretically transferred to PVDF (poluvinlidene difluoride) membrane (Millipore). The membrane was incubated with antibody against PCBP1 (1:1,000, ab168377, Abcam, USA) and β-actin (1:2,000, Santa Cruz Biotech, Santa Cruz, CA, USA) at 4°C overnight. After being washed, these membranes were followed by HRP (horseradish peroxidase)-labeled goat anti-rabbit immunoglobulin G (IgG) (1:2,000, Santa Cruz Biotechnology, USA) at room temperature for 2 h. The signals were visualized with enhanced chemiluminescence (ECL; Beyotime Company) and analyzed using a BI-2000 system. To quantify the protein band intensities, the films were analyzed using NIH ImageJ software.
6
Western Blot Analysis of Protein Expression
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Images of HEI-OC1 cells treated with different reagents were captured by optical microscope. Then, the total proteins of treated cells or tissues were extracted by RIPA lysis buffer (Thermo, 89901, USA), in which proteinase inhibitor (1:100, Selleck, TX, USA) was added. After the concentration measurements by BCA assay kit (Beyotime Biotechnology, Shanghai, China), equal amounts of protein were denatured and then separated by 12% SDS-PAGE electrophoresis, followed by transfer to polyvinylidene fluoride membranes (PVDF, Millipore, Darmstadt, Germany). The membranes were blocked in 5% non-fat milk for 1 h at room temperature. After washing with TBS containing 0.05% tween 20 (TBST) three times, the membranes were incubated with related primary antibodies (1:1,000) in TBST with 5% BSA overnight. Then, they were incubated with secondary antibodies (1:5,000–1:10,000) for 1 h after three washes with TBST. Finally, the protein signals were detected by use of the ECL kit (Millipore, WBKLS0010, Darmstadt, Germany) and analyzed by ImageJ software.
7
Cell Lysis and Immunoprecipitation Protocol
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Cells transfected with indicated plasmids were pelleted with 1% SDS and boiled in 100 °C for 10 min. These samples were then diluted in TNE buffer (50 mM Tris (pH 7.5), 150 mM NaCl, 1% NP-40, 1% Triton X-100, 0.5% sodium deoxycholate, 10 mM NaF, 1 mM Na3VO4 and proteinase inhibitor (Selleck)) with 0.1% SDS for subsequent immunoprecipitation. The lysates and immunoprecipitates were analyzed by immunoblotting.
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