For each cell type, 3 × 106 cells were lysed in ice-cold cell extraction buffer (Invitrogen, USA) containing 1 mM PMSF (Abcam, Cambridge, UK) and 20 μl/mL protease inhibitor cocktail (Sigma-Aldrich, Steinheim, Germany) for 30 min. The lysates were centrifuged at 13000 rpm for 10 min at 4 °C. The total protein concentration was determined by a Qubit® protein assay kit (Invitrogen, USA) using a Qubit 3.0 fluorometer (Invitrogen, USA). Cell lysates (30 μg) were separated by Bolt™ 4–12% Bis-Tris plus gels (Invitrogen, USA) and transferred to PVDF membranes (Millipore, USA). Proteins were detected using primary antibodies against BCAT2 (Abcam, Cambridge, UK) and GAPDH (Invitrogen, USA) and a WesternBreeze® chemiluminescent kit (Invitrogen, USA) according to the manufacturer’s instructions. Bands were visualized using the G:Box Chemi Gel Documentation system (Syngene, USA).
Phenyl methyl sulfonyl fluoride (pmsf)
Manufactured by Abcam
Sourced in United Kingdom, United States
PMSF is a protease inhibitor used in laboratory settings. It functions to inhibit the activity of serine proteases, which are enzymes that cleave peptide bonds in proteins. PMSF is commonly used in protein extraction and purification protocols to prevent proteolytic degradation of target proteins.
Automatically generated - may contain errors
Lab products found in correlation
G box chemi gel documentation system, by Syngene (2 mentions)
Western breeze chemiluminescent kit, by Thermo Fisher Scientific (2 mentions)
Qubit protein assay kit, by Thermo Fisher Scientific (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Bolt 4 12 bis tris plus gel, by Thermo Fisher Scientific (2 mentions)
Qubit 3.0 fluorometer, by Thermo Fisher Scientific (2 mentions)
Cell extraction buffer, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
Enhanced chemiluminescence, by Thermo Fisher Scientific (2 mentions)
Mouse monoclonal anti β actin antibody, by Merck Group (2 mentions)
Gapdh, by Thermo Fisher Scientific (1 mentions)
0.45 μm pvdf membrane, by GE Healthcare (1 mentions)
Ab99359, by Abcam (1 mentions)
Leupeptin, by Merck Group (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Enhanced chemiluminescent reagent, by Beyotime (1 mentions)
Ripa buffer, by Solarbio (1 mentions)
Bicinchoninic acid protein assay kit, by Thermo Fisher Scientific (1 mentions)
Enhanced chemiluminescence ecl detection reagent, by Thermo Fisher Scientific (1 mentions)
Horseradish peroxidase linked secondary antibody, by Jackson ImmunoResearch (1 mentions)
11 protocols using phenyl methyl sulfonyl fluoride (pmsf)
1
Quantification of BCAT2 Protein Levels
2
Western Blot Protein Analysis Protocol
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Cells were washed with PBS, then lysed with RIPA lysis buffer supplemented with 100 x protease inhibitors, 100 x phosphatase inhibitors and 0.1 M PMSF (Abcam, USA). Cell lysates were then harvested by centrifugation at 14000 rpm for 5 minutes, at 4°C. Protein extracts in the supernatant were transferred into new tubes, and protein concentration was measured by BCA protein assay method. Proteins were analyzed using 10% SDS-PAGE gel electrophoresis, and then transferred onto a PVDF membrane. The membrane was incubated in 5% skim milk in TBS-T for blocking at room temperature for 1 hour, washed with TBS-T three times for 15 minutes, and then incubated with primary antibody solution (1:1000 dilution for all antibodies) at 4°C over-night, followed by secondary antibody incubation for 3 hours at room temperature. The immuno-reactive signals were visualized with Super Signal West Pico Chemiluminescent Substrate, using G-BOX chemi-XRQ. Detected signals were quantified using Genesys. ß-actin proteins were shown as a loading control and used for quantification normalization.
3
Protein Expression Analysis in BCC Cells
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BCC cells (3 × 106) were lysed in ice-cold cell extraction buffer (Invitrogen, Carlsbad, CA, USA) supplemented with 20 μL/mL protease inhibitor cocktail (Sigma-Aldrich, Germany) and 1 mM PMSF (Abcam, Cambridge, U.K.) for 30 min. The lysates were centrifuged at 13,000 rpm for 10 min at 4 °C. Qubit® protein assay kit (Invitrogen) was used to determine total protein concentration by Qubit 3.0 fluorometer (Invitrogen). Cell lysates (30 μg) were separated by Bolt™ 4–12% Bis-Tris plus gels (Invitrogen) in MES SDS running buffer and transferred to 0.45 μm PVDF membranes (GE Healthcare, U.K.). Proteins were detected using primary antibodies against ECHS1 (ab174312) and FASN (ab99359) (Abcam), PPAR-δ (PA1-823A), ME1 (MA5-23524), ME2 (PA5-38007), ME3 (PA5-36494), and GAPDH (AM4300) (Thermo Fisher Scientific, USA), and a WesternBreeze® chemiluminescent kit (Invitrogen) according to the manufacturer’s instructions. Bands were visualized by G:Box Chemi Gel Documentation system (Syngene, Frederick, MD, USA).
4
Affinity Purification of Cysteine Cathepsins
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In order to pull down the in vitro labelled cysteine cathepsins, 250 μL (about 400 μg) of protein concentrations of cells extracts were measured according to Bradford 1976 [45 (link)]), and cellular extracts that were previously labelled with DCG04 were diluted with 750 μL binding buffer (20 mM sodium acetate, pH 5.5, 150 mM sodium chloride, 0.1% triton X-100, 10 μg/mL E-64, 10 μg/mL leupeptin (Sigma Aldrich, St. Louis, MO, USA), 1 mM PMSF (Abcam)), and the mixture was centrifuged at 14,000× g for five min, and supernatant was incubated overnight with 40 μL settled streptavidin beads at 4 °C. Beads were precipitated by centrifugation for 5 min at 3000 rpm, then they were washed five times with 20 mM sodium acetate (pH 5.5), 150 mM sodium chloride, and 0.1% triton X-100 and twice with 10 mM Tris-HCL, pH 6.8 [46 (link)]. Settled beads were mixed with 40 μL 2X sample buffer and heated five min at 95 °C [47 (link),48 (link),49 (link),50 (link)]. The supernatant was subjected to SDS-PAGE and blotting on nitrocellulose membrane and immunoblotted with antibodies (1:2000) (ThermoFischer Scientific, Waltham, MA, USA) against cathepsin B, D, and L [51 (link)].
5
Western Blot Analysis of Protein Signaling
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Protein was extracted from treated cells including Eca109 and KY-SE150 using RIPA buffer (Beijing Solarbio Science & Technology Co., Ltd.) with protease inhibitor (PMSF; Abcam) and quantified using a bicinchoninic acid protein assay kit (Thermo Fisher Scientific, Inc.). Equal amounts of protein (20 µg) were separated using 12% SDS-PAGE, and then transferred onto PVDF membranes. Subsequently, the membranes were incubated in 5% skimmed milk for 1 h at room temperature and then incubated with primary antibodies (Abcam), including SPATS2 (cat. no. ab122495; 1:500), cyclin E (cat. no. ab33911; 1:1,000), P53 (cat. no. ab32389; 1:1,000), matrix metalloproteinase (MMP-9; cat. no. ab73734; 1 µg/ml), N-cadherin (cat. no. ab202030; 1:500), and GAPDH (cat. no. ab181602; 1:10,000) at 4˚C overnight. After washing the membranes with TBS-Tween-20 (0.1%) three times, they were incubated with secondary antibody goat anti-rabbit IgG H&L (cat. no. ab205718; 1:2,000; Abcam) at room temperature for 1 h. After washing the membranes three times with TBS-T, the signals were detected using enhanced chemiluminescent reagent (Beyotime Institute of Biotechnology) and densitometry was estimated using Quantity One software (v4.6.6; Bio-Rad Laboratories, Inc.). GAPDH was used as a normalization control.
6
Western Blot Analysis of Spinal, Amygdalar, and Cortical Proteins
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All the animals were sacrificed under deep sevoflurane (3%) anesthesia. The spinal dorsal horn segments (L3–L5), amygdala and cortex were removed quickly and snap-frozen in liquid nitrogen. The tissues underwent mechanical homogenization in chilled RIPA buffer with PMSF (Abcam, Cambridge, UK). The lysates were cleared by centrifugation, and supernatants were obtained for total protein extraction. Protein amounts were assessed by the bicinchoninic acid assay. Protein separation was carried out by 10% SDS-PAGE. After transfer onto nitrocellulose membranes, the specimens underwent successive incubations with rabbit monoclonal antibodies targeting pentraxin-3 and rabbit polyclonal antibodies nectin-1 (1:1000; Santa Cruz, CA, USA), respectively, and horseradish peroxidase-linked secondary antibodies (1:2000, Jackson ImmunoResearch, PA, USA). Enhanced chemiluminescence (ECL) detection reagents (Thermo Scientific, IL, USA) were utilized for development, followed by quantification with Gene Tools Match software (Syngene, Cambridge, UK). Antibodies targeting β-actin (1:5000; Sigma, MO, USA) were utilized for normalization.
7
Spinal Dorsal Horn Protein Extraction and Analysis
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All the animals were sacrificed under deep anesthesia of sevoflurane (3%). The left L3-5 segments of spinal dorsal horn were removed rapidly and homogenized in ice-cold RIPA buffer containing PMSF (Abcam, Cambridge, UK). The lysate was centrifuged, and the supernatant was collected as the total protein. The protein content was determined using the bicinchoninic acid assay method. The equivalent amount of proteins was resolved on a 10% SDS-PAGE gel, transferred to nitrocellulose membrane and probed with monoclonal mouse anti-β-actin antibody (42 KDa; 1:5000; Sigma-Aldrich) and polyclonal rabbit antibody against transferrin receptor 1 (TfR1, 1:5000; ZenBioScience, Durham, NC, USA), followed by incubation with horseradish peroxidase-conjugated secondary antibodies (1:2000, Jackson ImmunoResearch, West Grove, PA, USA). The membrane-bound secondary antibodies were visualized with enhanced chemiluminescence (Thermo Scientific, Rockford, IL, USA) and quantified using Gene Tools Match software (Syngene, Cambridge, UK).
8
Western Blot Analysis of Apoptosis Regulators
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NIT-1 cells were lysed in 100 μL of modified RIPA protein lysis buffer supplemented with 1 × PMSF (Abcam, Cambridge, UK) in each well of the 6-well plates. The proteins were then separated by SDS-PAGE (Abcam, Cambridge, UK) and transferred onto nitrocellulose membranes (CST, Beverly, USA) without delay. Subsequently, the membranes were incubated with primary antibodies at 4 °C for 12 h, followed by appropriate secondary antibodies at room temperature for 2 h. Blots were visualized using a Tanon 5200 visualizer (Tanon, Shanghai, China). The antibodies employed in this experiment included mouse anti-METTL14 (ab220030, 1:1000, Abcam, Cambridge, UK), rabbit anti-Bcl-xL (2764, 1:1000, CST, Beverly, MA, USA), rabbit anti-Bim (2933, 1:1000, CST, Beverly, MA, USA), rabbit anti-cleaved caspase 3 (9664, 1:1000, CST, Beverly, MA, USA), with β-actin (T0022, 1:3000, Affinity, Cincinnati, OH, USA) serving as a reference for total protein levels. Data analysis was performed using ImageJ software.
9
Western Blot Analysis of Protein Expression
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After the indicated treatments, cells were harvested and lysed in cell lysis buffer (Beyotime Institute of Biotechnology, Haimen, China) supplemented with PMSF (Abcam, Cambridge, UK). The lysed samples were denaturated and then subjected to SDS-PAGE, followed by transferring onto polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, USA). The membrane was blocked with 5% nonfat dry milk for 1 h at room temperature and incubated with primary antibody overnight at 4 °C. And the corresponding secondary antibody was added for incubation at room temperature for 1h. Protein bands was detected by ECL Western Blotting Substrate (Solarbio, Beijing, China), visualized by Amersham ImageQuant 800 (Cytiva, Japan). Image J was used to analyze the relative protein levels.
Antibodies used in Western blot were anti-ACE2 (AF933, R&D Systems, 1 μg/mL); anti-SKP2 (2652S, Cell Signaling Technology, 1:1000); anti-β-tubulin (ab6046, Abcam, 1:4000); anti-GAPDH (AM1020b, Abcepta, 1:2000); Donkey Anti-Goat IgG H&L (HRP) (ab97110, Abcam, 1:1000); Goat Anti-Rabbit IgG H&L (HRP) (ab205718, Abcam, 1:1000); Anti-mouse IgG, HRP-linked Antibody (7076S, Cell Signaling Technology, 1:1000).
Antibodies used in Western blot were anti-ACE2 (AF933, R&D Systems, 1 μg/mL); anti-SKP2 (2652S, Cell Signaling Technology, 1:1000); anti-β-tubulin (ab6046, Abcam, 1:4000); anti-GAPDH (AM1020b, Abcepta, 1:2000); Donkey Anti-Goat IgG H&L (HRP) (ab97110, Abcam, 1:1000); Goat Anti-Rabbit IgG H&L (HRP) (ab205718, Abcam, 1:1000); Anti-mouse IgG, HRP-linked Antibody (7076S, Cell Signaling Technology, 1:1000).
10
Western Blot Analysis of Spinal Cord Proteins
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The animals were deeply anesthetized with sevoflurane (3%). The dorsal horns of the cervical (for ACD model) and lumbar (for BDL model) segments in the spinal cord were quickly removed and cryopreserved in liquid nitrogen. The sample was homogenized mechanically in ice-cold RIPA buffer that contained PMSF (Abcam, Cambridge, UK). The bicinchoninic acid test method was used to assess the amount of protein present. Using a membrane coated with a monoclonal mouse anti-β-actin antibody (1:5000; Sigma-Aldrich), the loading and blotting of an identical quantity of total proteins were confirmed. Following resolution on a 10% SDS-PAGE gel, the samples were transferred to nitrocellulose membranes, and probed with rabbit antibodies against ANXA1 (1:2000, ab137745, Abcam), transferrin receptor 1 (TfR1, 1:2000, ab214039, Abcam), iron regulatory protein 1 (IRP1, 1:2000, ab236773, Abcam) and divalent metal transporter 1 (DMT1, 1:2000, ab262715, Abcam). After which secondary antibodies coupled with horseradish peroxidase (1:2000, Jackson Immuno Research, West Grove, PA, USA) were incubated. Enhanced chemiluminescence (Thermo Scientific, Rockford, IL, USA) was used to visualize the membrane-bound secondary antibodies, and Media Cybernetics Inc.’s Image-Pro Plus software (Version 6.0) was used to quantify the results.
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