RIPA buffer (Beyotime, China) and the protease inhibitor PMSF (Solarbio, China) (RIPA: PMSF ratio = 100:1) were used to extract total proteins. The extracted proteins were centrifuged at 13,000 g and 4 °C for 10 min. The BCA protein assay kit (Solarbio, China) was used to quantify the protein content. An automatic protein analyzer (Wes & Jess, USA) was used for the Western blotting assay in accordance with the specific protocols, and β-actin was used as a reference. The blots were then incubated with rabbit anti-human antibodies against β-actin (CST, USA), EDIL3, VEGF, and VEGFR2 (all from Abcam, UK).
Phenyl methyl sulfonyl fluoride (pmsf)
Manufactured by Solarbio
Sourced in China, United States, Switzerland
PMSF is a protease inhibitor commonly used in biochemical research to prevent protein degradation. It irreversibly inhibits serine proteases by binding to the active site. PMSF is typically used in sample preparation to preserve the integrity of proteins and enzymes during analysis.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (104 mentions)
Ripa buffer, by Solarbio (59 mentions)
Ripa lysis buffer, by Solarbio (45 mentions)
Bca protein assay kit, by Solarbio (28 mentions)
Radioimmunoprecipitation assay (ripa), by Solarbio (24 mentions)
Bca protein assay kit, by Beyotime (24 mentions)
Gapdh, by Proteintech (19 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (18 mentions)
Bca kit, by Solarbio (14 mentions)
Phosphatase inhibitor, by Solarbio (14 mentions)
Pvdf membrane, by Solarbio (12 mentions)
Bca kit, by Thermo Fisher Scientific (12 mentions)
Ripa buffer, by Beyotime (11 mentions)
Ripa lysis buffer, by Beyotime (10 mentions)
Polyvinylidene fluoride (pvdf), by Merck Group (10 mentions)
β actin, by Proteintech (10 mentions)
Polyvinylidene difluoride membrane, by Merck Group (10 mentions)
Odyssey infrared imaging system, by LI COR (8 mentions)
Chemidoc xrs system, by Bio-Rad (8 mentions)
β actin, by Abcam (8 mentions)
436 protocols using phenyl methyl sulfonyl fluoride (pmsf)
1
Quantitative Protein Analysis by Western Blot
2
Protein Extraction and Western Blotting
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Total protein extracts were prepared using RIPA buffer (Cat. #: R0010, Solarbio) supplemented with 1× PMSF (Cat. #: P0100, Solarbio) and 1× phosphatase inhibitor cocktail (Cat. #: HY‐K0022, MedChemExpress LLC, Monmouth Junction, NJ, USA). After 30 min of lysis on ice with frequent tapping, cells and dissected mouse tissues were centrifuged at 14,000 × g at 4°C for 15 min. The supernatant was mixed with SDS‐PAGE loading buffer (Cat. #: WB‐0091, DING GUO) at a ratio of 4:1 and incubated at 100°C for 10 min. The sample was loaded onto a 10 or 12.5% Tris‐glycine gel (Cat. #: PG213, Epizyme) and blotted onto a 0.2‐μm polyvinylidene difluoride membrane (Cat. #: ISEQ00010, Immobilon). Membranes were blocked with 5% skimmed milk at room temperature for 2 h and incubated for 12–16 h with indicated primary antibodies (Table S1 ). After incubation with the secondary antibody at room temperature for 2 h on the next day, membranes were washed in 1× TBST medium and imaged by using an Amersham Imager 600 (GE Healthcare Bio‐Sciences AB).
3
Western Blot Analysis of EMT Markers
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Cells lysates were prepared by RIPA (Solarbio, China) with %1 PMSF (Solarbio, China). Protein samples was separated by 10% SDS-PAGE (Beyotime Biotechnology, China) and transferred onto PVDF membranes. The membranes were blocked in 5% skim milk in TBST at room temperature for 2 h and then incubated with primary antibodies at 4 C overnight. After 3 times washing with TBST, membranes were incubated with Goat anti-rabbit secondary antibodies (1:4,000 in TBST) at room temperature for 2h. After 3 times washing with TBST, the target proteins were visualized using enhanced chemiluminescence kit (Proteintech) according to the manufacturer's protocol. Antibodies against FOXM1 and GAPDH were purchased from Proteintech. WB-used antibodies against E-cadherin, N-cadherin, Vimentin, Snail, ZEB1 were included EMT-sampler Kit, which was purchased from Cell Signaling Technology.
4
Western Blot Analysis of EMT Markers
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The cells were harvested with radioimmunoprecipitation assay (RIPA) lysis buffer (Solarbio) containing phenylmethanesulfonyl fluoride (PMSF; Solarbio) and protease inhibitor cocktail (Solarbio). The lysates were removed through centrifugation at 12,000 × g for 15 min at 4°C. The cell protein lysates were quantified, separated through sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred onto a nitrocellulose membrane, and then blocked with 5% skim milk (BD Biosciences) prepared in 1× TBST with 0.05% Tween-20 (Solarbio). Subsequently, the membrane was incubated with specific primary antibodies, washed, and probed with horseradish-peroxidase-conjugated secondary antibody. Several antibodies such as E-cadherin (1:2,000 dilution, rabbit anti-human polyclonal antibody, #20874-1-AP; Proteintech, Wuhan, China), N-cadherin (1:6,000 dilution, rabbit anti-human polyclonal antibody, #22018-1-AP; Proteintech), Vimentin (1:2,000 dilution, rabbit anti-human polyclonal antibody, #10366-1-AP; Proteintech), and GAPDH (1:6,000 dilution, rabbit anti-human polyclonal antibody, #10494-1-AP; Proteintech) were used. Furthermore, the signals were detected using Molecular Imager® (Tanon, Shanghai, China).
5
Quantitative Western Blot Analysis of P4HB
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Total proteins were extracted by the incubation of cultured HK-2 cells with radioimmunoprecipitation assay (RIPA) buffer (P0013D, Beyotime) and adding 1 % phenylmethylsulfonyl fluoride (PMSF) (329-98-6, Solarbio) and quantified by Bio-RAD assays. The same amount of protein was separated by 10 % sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (SDS-PAGE), and proteins were transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were then blocked with 5 % skim milk and subsequently incubated with primary antibodies against P4HB (1:1,000 dilution, Cat.137,110, Abcam) and GAPDH (1:6,000 dilution, Cat. 60004-1-Ig, ProteinTech) overnight at 4°C. After washing, the proteins were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (1:6,000 dilution, Cat. SA00001-2, ProteinTech) for 1 h at room temperature. The bands were visualized using an enhanced chemiluminescence (ECL) system, and the signal intensity was quantitatively processed by ImageJ software.
6
Ee-As4S4-Induced Apoptosis Pathway
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After incubated with ee-As4S4, Kasumi-1 cells were collected and lysed in RIPA cell lysis buffer (Solarbio life sciences, Beijing, China) supplemented with 1 mM PMSF (Solarbio life sciences, Beijing, China) and protein phosphatase inhibitor (Applygen, Beijing, China). The lysates were then centrifuged at 12,000 rpm for 15 min at 4°C. The protein was quantified by a Pierce™ BCA Protein Assay Kit according to the manufacturer’s instructions. After denatured by boiling in loading buffer (Applygen, Beijing, China), an equal amount of protein (20 µg) was loaded and separated on 12% glycine SDS-PAGE gel, and transferred to polyvinylidene difluoride (PVDF) membranes (0.45 µm; Millipore, Merck, Darmstadt, Germany). Then the membrane was incubated with primary antibodies at 4°C overnight, including anti-β-actin, anti-pro-caspase 3, and anti-caspase 3 (Cell Signaling Technology, CST, Boston, MA, USA). The membrane was incubated with Horseradish Peroxidase (HRP)-conjugated secondary antibodies and visualized using an automatic chemiluminescence image analysis system (Tanon, Shanghai, China) with HRP substrate luminol reagent and peroxide solution (Merck, Darmstadt, Germany). The relative expression of the protein was analyzed using Image J software.
7
Quantifying Total Protein in Tissues
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The total protein content in tissues and cells was extracted using RIPA buffer (Solarbio, Beijing, China) supplemented with 1% PMSF (Solarbio). The quantification was completed with the help of the BCA Protein Assay Kit (Solarbio). We used SDS–PAGE gels prepared from the PAGE Gel Fast Preparation Kit (Epizyme, shanghai, China) to separate the total protein (20 μg). Isolates were immediately anchored onto nitrocellulose membranes (Millipore, Danvers, MA, USA). A blocking solution for 1 h at room temperature was necessary for more specific antibody binding. The blotted nitrocellulose membranes were incubated with the primary antibody, anti-AOC1 antibody (1:1000; ab231558; Abcam, UK), anti-TFR antibody (1:1000; ab214039; Abcam), anti-TF antibody (1:1000; ab277635; Abcam,), anti-FTH1 antibody (1:1000; ab75973, Abcam), and anti-SOX15 antibody (1:100; sc-166964; Santa Cruz, Dallas, TX) overnight at 4 °C. After washing with Tris-buffered saline with Tween, the nitrocellulose membranes were incubated with goat anti-rabbit IgG (IRDye® 800CW; 1:5000; ab216773; Abcam) or goat anti-mouse IgG (IRDye® 800CW). Anti-GAPDH antibody (1:1000; ab9485; Abcam) was used as a loading control. The Odyssey CLx Infrared Imaging System (Gene Company Limited, Hong Kong, China) was used to detect the target protein bands.
8
Western Blot Analysis of MC1R Expression
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Proteins of different tissue samples were extracted using RIPA lysis buffer (Beyotime, Shanghai, China) containing 1% PMSF (Solarbio, Beijing, China), and their levels were measured with the BCA protein assay kit (Solarbio, Beijing, China).
Thirty micrograms of protein were denatured in sample buffer and then electrophoresed on 10% SDS–PAGE. The PVDF membranes (Immobilin, Carrigtwohill, Ireland) were incubated with primary antibodies (1:400 dilution of MC1R antibody (Immunoway, Tennyson Pkwy Ste 250, Plano, USA); 1:1000 dilution of β-actin antibody (Cell Signaling Technology, Denvers, Massachusetts, USA) overnight at 4 °C after electrophoresis, transfer, and blockage. Next, a secondary antibody (1:4000 dilution of goat anti-rabbit (Immunoway, Tennyson Pkwy Ste 250, Plano, USA)) was added at room temperature for 1 h. Then, the immunoblots were marked using ECL (Solarbio, Beijing, China). Finally, based on densitometry and ImageJ Software, analyses were performed in triplicate, and MC1R expression was presented as the grayscale value of MC1R compared to the corresponding β-actin in tissue.
Thirty micrograms of protein were denatured in sample buffer and then electrophoresed on 10% SDS–PAGE. The PVDF membranes (Immobilin, Carrigtwohill, Ireland) were incubated with primary antibodies (1:400 dilution of MC1R antibody (Immunoway, Tennyson Pkwy Ste 250, Plano, USA); 1:1000 dilution of β-actin antibody (Cell Signaling Technology, Denvers, Massachusetts, USA) overnight at 4 °C after electrophoresis, transfer, and blockage. Next, a secondary antibody (1:4000 dilution of goat anti-rabbit (Immunoway, Tennyson Pkwy Ste 250, Plano, USA)) was added at room temperature for 1 h. Then, the immunoblots were marked using ECL (Solarbio, Beijing, China). Finally, based on densitometry and ImageJ Software, analyses were performed in triplicate, and MC1R expression was presented as the grayscale value of MC1R compared to the corresponding β-actin in tissue.
9
Western Blot Analysis of Apoptosis Regulators
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After washing in cold PBS twice, GCs were treated with RIPA lysis buffer (Beyotime, 243 Shanghai, China) containing 1.5% proteasome inhibitor (PMSF, Solarbio, Beijing, China) for 20 min to isolate total protein. The concentration of each protein sample was quantified by using a BCA Kit (Biosharp, Beijing, China). A total of 15 μg of protein from each sample was loaded on 15% polyacrylamide gel and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (GenScript, Nanjing, China). After separation by electrophoresis, the proteins were transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA). The membranes were blocked in 5% nonfat milk for 1 h and then incubated with primary antibodies for 8 h at 4 °C. The bound primary antibody was visualized with secondary antibody using an ECL detection reagent (Advansta, CA, USA) with a LAS2000 imaging system (GE Healthcare, Chicago, IL, USA). The β-tubulin protein level was used as the internal control. The primary antibodies used in the study were anti-Caspase 3 (CASP3) (diluted at 1:1,000; Proteintech, Wuhan, China), anti-FoxO1 (diluted at 1:1,000; CST, BMA, USA), and anti-β-tubulin (diluted at 1:2,000; Proteintech, Wuhan, China).
10
Protein Extraction and Quantification for Western Blot
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The transplanted tumor tissues were lysed by radio immunoprecipitation assay (RIPA, Beyotime, China) buffer containing phenyl methane sulfonyl fluoride (PMSF, Solarbio, China) with mild sonication. The concentrations of total proteins were measured by the BCA Protein Assay Kit (Beyotime, China). Equal amount of protein was subjected to 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) and blotted on polyvinylidene fluoride (PVDF, Millipore, Billerica, USA). Protein bands were visualized via enhanced chemiluminescence (ECL, Millipore, USA) and were analyzed using the western blot imaging system (AI600 images, GE, USA), followed by measurement of the density of each band using Image J software.
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