The HEp-2 cells were harvested and washed once with phosphate-buffered saline (PBS), and further lysed in radioimmunoprecipitation assay buffer with phenyl-methylsulfonyl fluoride (Sangon Biotech Co., Ltd.). Protein concentrations were determined using a Pierce BCA protein assay kit (Thermo Fisher Scientific, Inc.). SDS-PAGE (12% gel) was utilized to separate equal quantities (20 μg) of the proteins, which were then electrically transferred onto nitrocellulose membrane (GE Healthcare Life Sciences, Chalfont, UK), and blocked with 5% fat free milk in Tris-buffered saline with 0.05% Tween (TBST) buffer for 1 h at room temperature. Individual membranes were further incubated with appropriately diluted primary antibodies (1:1,000) overnight at 4°C. Horseradish peroxidase (HRP)-conjugated secondary antibodies (1:5,000) were then applied for 2 h at room temperature following three intensive washes in TBST. The HRP substrate gives rise to the final images using electrochemiluminescence (Western Bright kit; cat. no. R-03031-D25; Advansta, Inc., Menlo Park, CA, USA), visualized following X-film development [Fujifilm (China) Investment Co., Ltd., Shanghai, China].
Phenylmethylsulfonyl fluoride
Manufactured by Sangon
Sourced in China
Phenylmethylsulfonyl fluoride is a versatile lab reagent used for protease inhibition. It irreversibly inactivates a wide range of serine proteases, making it a valuable tool in various biochemical applications.
Automatically generated - may contain errors
Lab products found in correlation
Bca kit, by Thermo Fisher Scientific (3 mentions)
Irdye 680lt donkey anti rabbit igg secondary antibodies, by LI COR (2 mentions)
Anti β actin antibody, by Cell Signaling Technology (2 mentions)
Odyssey infrared imaging system, by LI COR (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Phosstop phosphatase inhibitor cocktail, by Roche (2 mentions)
Nitrocellulose membrane, by GE Healthcare (1 mentions)
Ripa lysis buffer, by Cell Signaling Technology (1 mentions)
Semi dry transfer system, by Bio-Rad (1 mentions)
Ab32047, by Abcam (1 mentions)
Anti p ampk antibody, by Cell Signaling Technology (1 mentions)
Anti ampk antibody, by Abcam (1 mentions)
Anti foxo1, by Cell Signaling Technology (1 mentions)
Anti bdnf sc 546, by Santa Cruz Biotechnology (1 mentions)
Lysis buffer, by Beyotime (1 mentions)
Aprotinin, by Sangon (1 mentions)
Rat fibrinogen, by Merck Group (1 mentions)
Thrombin, by Merck Group (1 mentions)
Pepstatin, by Sangon (1 mentions)
5 protocols using phenylmethylsulfonyl fluoride
1
Western Blot Protocol for Protein Analysis
2
Mitochondrial Protein Expression Analysis
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Total proteins from liver tissue were extracted using RIPA lysis buffer (Cell Signaling Technology, USA) supplemented with the protease inhibitor phenylmethylsulfonyl fluoride (1 mM; Sangon Biotech, Shanghai, China) and boiled for 5 min at 95 °C. Proteins separated using BN-PAGE or SDS-PAGE were transferred to 0.22 mm polyvinylidene fluoride membranes (Bio-Rad, Hercules, CA) using a semidry transfer system (Bio-Rad) and probed with the following primary antibodies: anti-MTCO1 (ab14705; 1:1,000), anti-COX2 (376,731; 1:2,000), anti-mfn1 (ab126575; 1:1,000), anti-mfn2 (ab260861; 1:1,000); anti-DRP1 (ab184247; 1:1,000), anti-OPA1 (ab157457; 1:1,000), anti-HSP60 (ab190828; 1:1,000), anti-CLPX (ab168338; 1:1,000), anti-GRP75 (sc133137; 1:1,000), and anti-CLPP (ab124822; 1:1,000). Membranes were incubated with primary and secondary antibodies. The expression of each protein was compared with that of GAPDH (sc-365062; 1:2000).
3
Protein Extraction and Western Blot Analysis
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Total proteins were extracted as previously described.23 , 26 , 27 The tissues were briefly homogenized in radioimmunoprecipitation assay (RIPA) lysis buffer (Biotime) supplemented with 1% phenylmethylsulfonyl fluoride (Sangon Biotech) and 1× PhosSTOP phosphatase inhibitor cocktail (Roche Applied Science). Protein concentration was measured using a butyleyanoacrylate (BCA) kit according to the manufacturer's instructions (Thermo Scientific). The proteins were separated using SDS‐polyacrylamide gel electrophoresis (SDS‐PAGE) and then transferred onto a polyvinylidene fluoride (PVDF) membrane (Millipore). The membranes were blocked in 5% bovine albumin (BSA) with a TBST buffer (20 mM Tris–HCl, pH 7.4, 150 mM NaCl, 0.1% Tween 20), followed by incubation with primary antibodies diluted in TBST containing 2.5% BSA at 4°C overnight: anti‐PPM1F (1:500, PA5‐15571, Invitrogen), anti‐p‐AMPK antibody (1:1000, #2535, Cell Signaling Technology), anti‐AMPK antibody (1:1000, ab32047, Abcam), and anti‐β‐actin antibody (1:1000, 4970, Cell Signaling Technology). Then, the membranes were washed with 1× TBST and incubated with IRDye 680LT donkey anti‐rabbit IgG secondary antibodies (1:5000, 926–68,023, Li‐COR Biosciences). Fluorescence was visualized and analyzed using an Odyssey infrared imaging system (Li‐COR Biosciences).
4
Western Blot Analysis of BDNF and FoxO1
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Western blots were performed as described previously [43 ]. Briefly, the dissected mPFC samples were homogenized in a lysis buffer (Beyotime Biotechnology, Shanghai, China) with 1% phenylmethylsulfonyl fluoride (Sangon Biotech, Shanghai, China) and 1 × PhosSTOP phosphatase inhibitor cocktail (Roche Applied Science, Penzberg, Germany). The extracted proteins were separated on a 15% SDS-PAGE gel and transferred to PVDF membrane (Millipore, Massachusetts, USA). The membrane was blocked with 5% non-fat milk powder in TBST buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% Tween20), followed by incubation with the following primary antibodies diluted in a blocking solution overnight at 4 °C: anti-BDNF (sc-546, 1:500, Santa Cruz Biotechnology, Dallas, TX, USA), anti-FoxO1 (1:500, #2880, Cell Signaling Technology, Danvers, MA, USA), or anti-β-actin antibody (1:1000, #4970, Cell Signaling Technology, Danvers, MA, USA). After washing, the membrane was incubated with IRDye 680LT donkey anti-rabbit IgG secondary antibodies (1:5000; 926–68,023, Li-COR Biosciences, Lincoln, NE, USA). The fluorescence was visualized and analyzed using an Odyssey Infrared Imaging System (Li-COR Biosciences, Lincoln, NE, USA).
5
Soybean Milk Protease Inhibition
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Soybean and milk were purchased from a local market. Milk fermented with Lactobacillus bulgaricus and Streptococcus thermophilus was obtained from Bright Dairy & Food Co. Enzyme inhibitors, including phenylmethylsulfonyl fluoride (PMSF; an inhibitor of serine protease and cysteine protease), phosphoramidon (an inhibitor of metallopeptidase), aprotinin (an inhibitor of trypsin and chymotrypsin), pepstatin (an acid protease inhibitor), and E-64 (a cysteine protease inhibitor) were purchased from Sangon Biotech Co. Ltd. The rat fibrinogen and thrombin were purchased from Sigma-Aldrich. All other chemical reagents were obtained from Sinopharm Chemical Reagent Co. Ltd.
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