Total protein from HUVECs was extracted using RIPA lysis buffer containing 2% protease inhibitor and 2% protease inhibitor (Beyotime, Shanghai, China). The total amount of protein in the lysate was measured by standard micro bicinchoninic acid (BCA) analysis according to the manufacturer's instructions. The samples were prepared in 5 × SDS-PAGE Sample Loading buffer and heated at 95 °C for 10 min before loading onto the gel. Protein samples and the protein marker (20 μL samples per lane) were separated on a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) running gel at 120 V for 50 min. Then the proteins were transferred onto PVDF membranes at 100 V for 120 min. Blocking was performed in 5% non-fat milk for 2 h. The blots were incubated with primary antibodies overnight on a shaking table at 4 °C. The membranes were then rinsed thoroughly in TBST and subsequently incubated with horseradish peroxidase–conjugated goat anti-rabbit or anti-mouse antibodies at 1:5000 for 90 min at room temperature. Finally, the signals were detected with ECL Super Signal West Pico Chemiluminescent Substrate, visualized using the Gelview 6000Plus Image Capture System (Guangzhou Biolight Biotechnology Co., Ltd, Guangzhou, China), and quantified by gel analysis using ImageJ software.
Protease inhibitor
Manufactured by Beyotime
Sourced in China, United States, Switzerland
Protease inhibitors are chemical compounds that block the activity of proteases, which are enzymes that break down proteins. They play a crucial role in various biological processes and have applications in research, diagnostics, and therapeutic fields.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (125 mentions)
Ripa buffer, by Beyotime (115 mentions)
Ripa lysis buffer, by Beyotime (108 mentions)
Bca protein assay kit, by Beyotime (76 mentions)
Phosphatase inhibitor, by Beyotime (47 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (40 mentions)
Polyvinylidene difluoride membrane, by Merck Group (30 mentions)
Gapdh, by Proteintech (28 mentions)
Bca kit, by Beyotime (22 mentions)
Bca assay kit, by Beyotime (21 mentions)
Radioimmunoprecipitation assay (ripa), by Beyotime (21 mentions)
Polyvinylidene fluoride membrane, by Merck Group (18 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (16 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (15 mentions)
β actin, by Proteintech (15 mentions)
β actin, by Abcam (14 mentions)
Bca kit, by Thermo Fisher Scientific (14 mentions)
Image lab software, by Bio-Rad (12 mentions)
Gapdh, by Cell Signaling Technology (12 mentions)
Ab6721, by Abcam (11 mentions)
683 protocols using protease inhibitor
1
Protein Extraction and Western Blot Analysis
2
Profiling lnc-AROD Interactions by AGO2 IP and MS2-RIP
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For AGO2 immunoprecipitation, lnc-AROD-overexpressing 293T cells were plated into six-well plates in three replicates and transfected with 3×Flag-AGO2 or 3×Flag-GFP for 48 h. The cells were UV cross-linked and lysed with RIPA buffer (10 mM HEPES [pH 7.4], 200 mM NaCl, 30 mM EDTA, and 0.5% Triton X-100) supplemented with RNase (Beyotime, China) and protease inhibitors for 15 min. The lysates were incubated with magnetic beads conjugated with IgG (as a control) and an anti-Flag antibody (F1804; Sigma-Aldrich, St. Louis, MO, USA) at 4°C for 4 h. The beads were washed five times and lysed in TRIzol reagent for RNA extraction and subjected to RT-qPCR to determine the amount of lnc-AROD.
An MS2-RIP assay was performed as previously reported (47 (link)). Briefly, 293T cells were plated into six-well plates in three duplicates and transfected with MS2 or MS2–lnc-AROD together with MS2-GST and miR-324-5p for 48 h. The cells were subsequently lysed with NT2 buffer (50 mM Tris-HCl [pH 7.0], 150 mM NaCl, 1 mM MgCl2, 0.05% NP-40, 1 mM phenylmethylsulfonyl fluoride, 10 mM ribonucleoside vanadyl complex) supplemented with RNase (Beyotime, China) and protease inhibitors for 15 min. The lysates were incubated with GST-magnetic beads at 4°C for 4 h and washed five times, and RNA was extracted from the beads followed by RT-qPCR to quantify the amount of miR-324-5p.
An MS2-RIP assay was performed as previously reported (47 (link)). Briefly, 293T cells were plated into six-well plates in three duplicates and transfected with MS2 or MS2–lnc-AROD together with MS2-GST and miR-324-5p for 48 h. The cells were subsequently lysed with NT2 buffer (50 mM Tris-HCl [pH 7.0], 150 mM NaCl, 1 mM MgCl2, 0.05% NP-40, 1 mM phenylmethylsulfonyl fluoride, 10 mM ribonucleoside vanadyl complex) supplemented with RNase (Beyotime, China) and protease inhibitors for 15 min. The lysates were incubated with GST-magnetic beads at 4°C for 4 h and washed five times, and RNA was extracted from the beads followed by RT-qPCR to quantify the amount of miR-324-5p.
3
STAT3 Interaction Analysis by Co-IP
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Cells or tumor tissue were lysed with RIPA buffer (Beyotime, Shanghai, China) supplemented with protease inhibitors (Beyotime, Shanghai, China) and phosphatase inhibitors (Bimake, USA). Lysates were denatured by heating for 5 min at 99 °C and loaded on 4-10% SDS-polyacrylamide gel electrophoresis. Then the proteins were transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, USA). The membranes were blocked and probed with primary antibodies and secondary HPR-conjugated antibodies. At last the membranes were detected by chemiluminescence (Tanon, Shanghai, China). For co-immunoprecipitation experiments, 293T cells were plated and grew to about 80% confluency, then co-transfected with plasmids of HA-STAT3 and Flag-STAT3 using Lipofectamine 2000. After 24 h, cells were treated with different concentrations of W2014-S for following 24 h, and stimulated with IL-6 (100 ng/mL) for 1 h. Cells were lysed with IP RIPA buffer (Beyotime, Shanghai, China) supplemented with protease inhibitors (Beyotime, Shanghai, China) and phosphatase inhibitors (Bimake, America), and lysates were cultured with Anti-flag Affinity Gel (B23102, Bimake, USA) overnight at 4 ℃. The gel was washed by PBST for three times and added 1×loading buffer and denatured by heating for 5 min at 99 ℃. Then the proteins were resolved on SDS-PAGE, transferred to PVDF membranes and analysed with immunoblotting.
4
Protein Extraction and Western Blot Analysis
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For animal tissues, after 3 days of modeling the skin wound, the wounded skin area and surrounding 5 mm skin tissues were removed from the skin, homogenized in RIPA buffer containing protease inhibitors (Beyotime, China), and proteins were extracted. For BMDMs cultivated in high-glucose DMEM in vitro, cells at the bottom of the dish and dead cells in the supernatant were collected after stimulation with LPS/ATP and different concentrations of ApoEVs. The proteins were extracted with RIPA buffer containing protease inhibitors (Beyotime, China). All proteins were loaded onto sodium dodecyl sulfate–polyacrylamide (SDS) gels and were transferred to polyvinylidene fluoride (PVDF) membranes following BCA quantification (Milipoll, USA). The membranes were blocked with 5% bovine serum albumin for 2 h at room temperature, treated overnight at 4 °C with primary antibodies, and then incubated for 2 h at room temperature with peroxidase-conjugated secondary antibodies (CWBio, China). Protein bands were detected with an imaging system (Tanon, China) and quantified with Image J software. The main antibodies included β-actin (CWBio, CW0096, China), GAPDH (CWBio, CW0100, China), NLRP3 (Abcam, ab270449, UK), caspase-1 (Abcam, ab138483, UK), cleaved caspase-1 (CST, 89332s, USA), and GSDMD (Abcam, ab209845, UK).
5
Chromatin Immunoprecipitation Assay for p63 Binding
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Chromatin immunoprecipitation (ChIP) assay was performed using Pierce Magnetic ChIP kit (ThermoFisher Scientific, Waltham, USA) according to the previous report
[28] . Cells were cross-linked by addition of formaldehyde (1% final concentration) to attached cells, rocked in 50 mM HEPES buffer (pH 7.5) containing 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100, and protease inhibitors (Beyotime), and then lysed in 10 mM Tris buffer (pH 8.0) containing 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, and protease inhibitors (Beyotime). Chromatin was collected, dialyzed against TE buffer and pre-cleared with a mixture of protein A and protein G Sepharose. Pre-cleared chromatin was incubated with 2 μg of anti-p63 antibody overnight at 4°C. Immunoprecipitates were washed and pellets were resuspended in 100 μL of TE and incubated at 55°C for 3 h. Cross-links were reversed by incubating samples at 65°C overnight, and samples were then extracted with phenol:chloroform and precipitated with ethanol. Pellets were resuspended in 100 μL of H
2O and subject to PCR analysis. Negative control IgG was also used in ChIP reaction.
[28] . Cells were cross-linked by addition of formaldehyde (1% final concentration) to attached cells, rocked in 50 mM HEPES buffer (pH 7.5) containing 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100, and protease inhibitors (Beyotime), and then lysed in 10 mM Tris buffer (pH 8.0) containing 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, and protease inhibitors (Beyotime). Chromatin was collected, dialyzed against TE buffer and pre-cleared with a mixture of protein A and protein G Sepharose. Pre-cleared chromatin was incubated with 2 μg of anti-p63 antibody overnight at 4°C. Immunoprecipitates were washed and pellets were resuspended in 100 μL of TE and incubated at 55°C for 3 h. Cross-links were reversed by incubating samples at 65°C overnight, and samples were then extracted with phenol:chloroform and precipitated with ethanol. Pellets were resuspended in 100 μL of H
2O and subject to PCR analysis. Negative control IgG was also used in ChIP reaction.
6
Protein Expression Analysis in LF Tissue
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A mixture of M-PER Tissue Protein Extraction Reagent (Pierce, IL, USA), EDTA, and protease inhibitors (Beyotime, Shanghai, China) was used to extract total protein from LF tissue samples, whereas LF cellular protein was isolated using RIPA lysis buffer containing protease inhibitors (Beyotime). A BCA kit (Beyotime) was then used to quantify protein levels in individual samples. Equal amounts of total protein from each sample (40 μg) were then separated via 10% SDS-PAGE and transferred onto PVDF membranes. Blots were then blocked with 5% SA, followed by overnight incubation with primary antibodies at 4° C. Antibodies were from Abcam (United Kingdom), were prepared in 5% BSA, and were as follows: anti-SIRT6 (#ab62739, 1:2000); anti-TGF-β1(#ab215715, 1:1000); anti-α-SMA (#ab7817, 1:3000); anti-Collagen I (#ab260043, 1:5000); anti-α-tubulin (#ab7291, 1:1000); and anti-β-actin (#ab8226, 1:5000). Following an additional 1 h incubation with HRP-conjugated secondary antibodies (#ab6789, 1:2000; #ab205718, 1:50000), protein bands were detected with an enhanced chemiluminescence kit (Millipore, Germany). The ImageJ software was then used for densitometric quantification, with α-tubulin or β-actin being used for normalization purposes.
7
Quantification of Inflammatory Cytokines in Tissues
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Tissue (50 ± 0.25 mg) was dissolved in 500 μL 1× protease inhibitor (Beyotime Biotechnology, Shanghai, China), which was grinded in a grinder (65 Hz, 45 s, −20 °C). The supernatant was collected after centrifugation at 4 °C at 13,000× g for 5 min. The inflammatory cytokines MCP-1, IL-1β, IL-6 and TNF-α in liver and epididymal fat were determined using commercially available ELISA kits (Solarbio life sciences Co., Ltd., Beijing, China) according to the manufacturers’ instructions. Total protein content in the liver and epididymis fat was measured with a bicinchoninic acid assay (BCA) protein assay kit (Sangon Biotech Co., Ltd., Shanghai, China). The levels were calculated by a method using Equation (2):
where O represents the relative values of inflammatory factors and P and Q represent the values of inflammatory factors and total protein content, respectively.
where O represents the relative values of inflammatory factors and P and Q represent the values of inflammatory factors and total protein content, respectively.
8
Western Blot Analysis of Protein Samples
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For WB analysis, cells were lysed in RIPA buffer (Beyotime Biotechnology, China) supplemented with 1 mmol/L phenylmethanesulfonyl fluoride (Beyotime Biotechnology) and 1× protease inhibitor (Beyotime Biotechnology). The cell lysates were centrifuged, and the pelleted debris was discarded. Total protein was then mixed with 5× SDS‐PAGE loading buffer and heated to 100°C for 5 min to prevent protein degradation. Protein samples (30 μg) were run on an SDS‐PAGE gel and then transferred to a PVDF membrane. Subsequently, the membrane was blocked with 5% milk for 1 h and then incubated with the indicated primary antibody overnight. After being washed with TBS Tween (TBST) solution, the membrane was incubated with a secondary antibody and detected with enhanced chemiluminescence (ECL) reagents. Relative protein expression was analyzed using ImageJ software.
9
Western Blot Analysis of TM-4 Cell Proteins
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TM-4 cells were lysed in RIPA buffer (Beyotime, China) with 1% protease inhibitor (Beyotime, China). Samples were separated by electrophoresis and transferred to PVDF membranes (Millipore, USA). Then, the samples were incubated with primary antibodies (Table 1 ) at 4° C overnight and incubated with secondary HRP-conjugated antibodies the next day for 1 h at room temperature (RT). The images were detected using an enhanced chemiluminescent kit (Beyotime, China). The bands were quantified using ImageJ.
10
Protein Interaction Analysis via Co-Immunoprecipitation
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Cells were lysed in lysis buffer (Beyotime, China) mixed with 1% protease inhibitor (Beyotime, China) to extract total protein. Lysates were centrifuged at 13,000 rpm for 10 min at 4°C to pelletize the cell debris. Protein concentration was measured using the BCA protein assay kit (Beyotime, China). Coimmunoprecipitation (co-IP) was performed using protein A-coated sepharose beads following the manufacturer's instructions. Briefly, freshly prepared precleared lysates were incubated overnight at 4°C with anti-IP3R1 (1 : 500, Invitrogen, UK) or anti-VDAC1 (1 : 1000, Abcam, UK) antibodies. Protein A beads were added and incubated for 2 h at 4°C, with rocking. The proteins pulled down by anti-IP3R1 or anti-VDAC1 antibodies were analyzed by western blotting [19 (link)].
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