Cells were washed with cold PBS and homogenized in lysis buffer containing protease inhibitors (keyGEN, Nanjing, China) on ice. After centrifugation, the protein-containing supernatant was collected. Total protein and 5x SDS loading buffer were mixed and boiled at 100°C for 5 min. Samples were separated by electrophoresis on 10% SDS-polyacrylamide gel and transferred onto polyvinylidene fluoride membranes, after which the membranes were blocked for 1 h at room temperature with 5% skim milk supplemented with 0.1% Tween 20 (TBST). Each membrane was then first incubated overnight with a primary antibody at 4°C and then with a secondary antibody for 120 min at room temperature. Immunoreactive bands were visualized using a chemiluminescence (ECL) detection system.
Protease inhibitor
Manufactured by Keygen Biotech
Sourced in China
Protease inhibitors are a class of laboratory reagents used to prevent the activity of proteolytic enzymes, known as proteases. These enzymes are responsible for breaking down proteins. Protease inhibitors are widely used in biochemical and cell biology research to maintain the integrity of protein samples by inhibiting unwanted proteolysis.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (13 mentions)
Phosphatase inhibitor, by Keygen Biotech (12 mentions)
Ripa buffer, by Keygen Biotech (4 mentions)
Ripa lysis buffer, by Keygen Biotech (4 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Phenylmethylsulfonyl fluoride, by Keygen Biotech (4 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Keygen Biotech (3 mentions)
Immobilon p, by Merck Group (2 mentions)
Gapdh, by Cell Signaling Technology (2 mentions)
Bca protein assay kit, by Keygen Biotech (2 mentions)
Pierce ip lysis buffer, by Thermo Fisher Scientific (2 mentions)
Ez ecl, by Sartorius (2 mentions)
Beyoecl plus, by Beyotime (2 mentions)
Gapdh, by Proteintech (2 mentions)
Western light, by Thermo Fisher Scientific (1 mentions)
Enhanced chemiluminescence reagent, by Bio-Rad (1 mentions)
Anti bax, by Abcam (1 mentions)
Anti caspase 3, by Abcam (1 mentions)
Anti bcl 2, by Abcam (1 mentions)
Anti p53, by Abcam (1 mentions)
39 protocols using protease inhibitor
1
Western Blot Protein Detection
2
Protein Extraction and Western Blot
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After cell attachment, the different conditioned media was collected. Tumor samples were minced on ice in pre-chilled lysis buffer containing phenylmethylsulfonyl fluoride, protease inhibitors, and phosphatase inhibitors (KeyGen Biotech, China). Homogenized tissues and cell lysates were then centrifuged at 14,000 rpm at 4°C for 15 min. Immunodetection was performed using a Western-Light chemiluminescent detection system (Applied Biosystems, Foster City, CA, United States) after incubation with the secondary antibody for 1 h.
3
Western Blot Protein Extraction Protocol
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GC cells were washed with low‐temperature PBS and then homogenized in lysis buffer containing protease inhibitors (KeyGEN, Nanjing, China) on ice. After centrifugation, the supernatant containing the protein was collected. A 4 × SDS loading buffer was added to the supernatant containing total protein and boiled at 100°C for 10 min. Protein samples were separated on 10–15% SDS‐polyacrylamide gel using electrophoresis and then transferred onto polyvinylidene fluoride membranes, which were subsequently blocked for 1.5 h at room temperature with 5% skim milk supplemented with 0.1% Tween 20 (TBST). Each of them was incubated 20 h with a primary antibody (1:1000) at 4°C and then with a secondary antibody for 60 min at room temperature. A chemiluminescence (ECL) detection system was utilized to visualize the immunoreactive bands.
4
Comprehensive Protein Analysis by Western Blot
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The cell extracts were prepared using RIPA buffer (KeyGen Biotech) containing protease inhibitors (KeyGen Biotech). Equal amounts of protein samples were subjected to 12% SDS-PAGE and transferred to PVDF membranes (Immobilon-P; Millipore, Billerica, USA). The membranes were then blotted with primary antibodies overnight at 4°C, followed by incubation with the HRP-conjugated goat anti-rabbit IgG secondary antibody (1:5000). The antibodies used were as follows: anti-AIFM2 (1:1000; Biorbyt, Cambridge, UK), anti-Bcl2 (1:2000; Abcam), anti-Bax (1:5000; Abcam); anti-Caspase3 (1:5000; Abcam), anti-Bak (1:1000; CST), anti-Cytc (1:1000; Abcam), anti-p53 (1:2000; Abcam), anti-p-mTOR (1:1000; CST), mTOR (1:1000; CST), anti-p-PI3K (1:1000; Affinity Biosciences), anti-PI3K (1:1000; Affinity Biosciences), anti-p-AKT (1:1000; CST), anti-AKT (1:1000; CST), anti-p-JAK2 (1:1000; Abcam), anti-JAK2 (1:1000; Abcam), anti-p-STAT3 (1:5000; Abcam), anti-STAT3 (1:5000; Abcam) and anti-GAPDH (1:5000; BBI, Shanghai, China). The protein bands were then visualized using enhanced chemiluminescence reagent (Bio-Rad, Hercules, USA). Band quantification was conducted using ImageJ (National Institutes of Health, Bethesda, USA).
5
Western Blot for Protein Detection
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HCT 116 and FHC cells were lysed with RIPA solution (Beyotime Biotechnology, Shanghai, China) containing protease inhibitors (Keygen, Nanjing, China) after treatment with appropriate drugs. Total protein was collected by centrifugation at 14,000× g for 15 min. The protein samples were separated by 10% sodium dodecyl sulfate (SDS) -polyacrylamide gel electrophoresis (PAGE) at 80 V and then transferred to a polyvinylidene fluoride (PVDF) membrane (Bio-RAD Laboratories). The membrane was then incubated with primary and secondary antibodies to detect target proteins. In the final step, the membrane was visualized using a chemiluminescence detection kit (Pierce Biotechnology, Rockford, IL, USA) according to the manufacturer’s instructions. The target protein was standardized as glyceraldehyde -3-phosphate dehydrogenase (GAPDH) and used as a sample control.
6
Protein Expression Analysis After Ischemic Injury
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Following reperfusion, ipsilateral brain tissue was homogenized in RIPA lysis buffer containing protease inhibitors (Nanjing KeyGen Biotech Co., Ltd.). The protein concentration of the supernatants (24,148.8 × g, 5 min, 4°C) was measured using a BCA Protein Quantitation Assay (Nanjing KeyGen Biotech Co., Ltd.). Equal amounts of protein (30 μg) were loaded onto 8-10% sodium dodecyl sulfate-polyacrylamide gels. Following electrophoresis, bands were transferred onto PVDF membranes. After being blocked with 5% BSA (OriGene Technologies, Inc.) for 2 h at 37°C, the membranes were incubated overnight at 4°C using the aforementioned primary antibodies against claudin-5, ZO-1, occludin, laminin and collagen IV or TNF-α (1:1,000; cat. no. 17590-1-AP; ProteinTech Group, Inc.), IL-1β (1:500; cat. no. ab200478; Abcam) or IL-6 (1:2,000; cat. no. 66146-1-Ig; ProteinTech Group, Inc.). The membranes were washed and incubated with HRP-conjugated secondary antibody (1:2,000; cat. no. SA00013-2; ProteinTech Group, Inc.) for 2 h at room temperature. Visualization was performed using ECL kit (cat. no. 34577; Thermo Fisher Scientific, Inc.). The immunoblots were visualized using a computerized image analysis system (Amersham Imager 600; Cytiva), and the results were expressed as the ratio of corresponding protein to β-actin (1:5,000; cat. no. SA00001-7L; ProteinTech Group, Inc.).
7
Protein Expression Analysis in HCC Cells
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HCC cells were suspended in serum-free media on a six-well plate at a density of 2.5 × 105/ml. A total of 2 ml of cell suspension was utilized, containing different concentrations of drug or inhibitor. After cell attachment, the conditioned media were collected. Tumor samples were minced on ice in prechilled lysis buffer containing phenylmethylsulfonyl fluoride, protease inhibitors, and phosphatase inhibitors (KeyGen BioTech, China). The homogenized tissues and cell lysates were then centrifuged at 14,000 rpm at 4°C for 15 min. The BCA Protein Assay Kit (Pierce Biotechnology, Rockford, IL) was employed to determine the total protein density and to ensure that equal amounts of proteins were separated on the 10% SDS-PAGE gel and transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, Boston, MA). After blocking with 5% nonfat milk for 1 hr. The membrane was incubated with the designated primary antibody at 4°C overnight. Immunodetection was performed using the Western-Light chemiluminescent detection system (Applied Biosystems, Foster City, CA) after incubation with the secondary antibody for 1 hr.
8
Quantifying MIP-3α in Caco2 and Colitis Models
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Cytokine chips were used to detect cytokines in Caco2 cell culture supernatants according to the manufacturer’s protocol. MIP-3α in culture supernatants from human Caco2 cells using a commercial ELISA kit (Lianshuo Biological, Shanghai, China) according to the manufacturer’s instructions.
For the expression of MIP-3α in the TNBS-induced colitis model, full-thickness colonic tissue specimens of mice were homogenised in PBS containing a cocktail of protease inhibitors (KeyGEN Bio TECH, Nanjing, China) supplemented with 1 mM PMSF. After centrifugation at 12,000 g for 10 min at 4 °C, the total protein level of the supernatants was determined with the BCA protein assay (Thermo Scientific, Rockford, IL, USA). To measure MIP-3α, a mouse double-antibody sandwich ELISA kit (R&D Systems, USA) was developed as described40 (link). The absorbance was measured at 450 nm and compared with the respective standard curve of the cytokines.
For the expression of MIP-3α in the TNBS-induced colitis model, full-thickness colonic tissue specimens of mice were homogenised in PBS containing a cocktail of protease inhibitors (KeyGEN Bio TECH, Nanjing, China) supplemented with 1 mM PMSF. After centrifugation at 12,000 g for 10 min at 4 °C, the total protein level of the supernatants was determined with the BCA protein assay (Thermo Scientific, Rockford, IL, USA). To measure MIP-3α, a mouse double-antibody sandwich ELISA kit (R&D Systems, USA) was developed as described40 (link). The absorbance was measured at 450 nm and compared with the respective standard curve of the cytokines.
9
Hippocampus Tissue Extraction and Analysis
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After finishing all behaviour tests, half of the mice in each group (n = 4) were anaesthetized and perfused intracardially with cold normal saline. The brains used for experiments were immediately isolated. And the hippocampus tissue was dissected out by using a dissecting microscope and homogenized in RIPA buffer (KeyGEN) with protease inhibitors (Key-GEN) and centrifuged at 12000 rpm for 10 min. The BCA kit (Solarbio) was used to determine the concentration of the hippocampus extract. The remaining mice (n = 4) were perused with saline and 4% paraformaldehyde (PFA) in phosphate buffer (PBS, 0.01 M, pH = 7.4). The brains were collected and frozen in liquid nitrogen. Consecutive coronal sections were sliced using a cryostat microtome (Leica) at 10 µm.
10
Western Blot Analysis of Cellular Proteins
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Total cellular protein was extracted and lysed with RIPA lysis buffer with protease inhibitors (KeyGEN, Nanjing, China) and phosphatase inhibitors (KeyGEN, Nanjing, China). Proteins were separated with 10% SDS-PAGE gels and transferred to PVDF membranes (Merck Millipore, MA, USA). Membranes were blocked with 5% non-fat milk for 1h and then incubated with primary antibodies of PPARγ, ANGPTL4, MMP-2, IL-1β, GAPDH, and tubulin β (Bioword, Beijing, China) overnight at 4 °C. After being washed five times for five minutes, the membranes were incubated with the corresponding secondary antibodies for 1h at room temperature. The protein bands were visualized with an Enhanced Chemiluminescence (ECL) detection kit (Amersham, NJ, USA). The protein bands were analyzed using Image Lab Software (Bio-Rad, CA, USA).
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