Components for culture media, tryptone, and yeast extract were purchased from Difco Laboratories, India. Glycine, phenylmethylsulfonyl fluoride (PMSF), isopropyl β-D-1-thiogalactopyranoside (IPTG), Tris buffer and sodium dodecyl sulfate (SDS) were from Amresco, United States. Glucose and NaCl were purchased from Qualigen, India. Low molecular weight marker for SDS-PAGE was from GE Healthcare, United States. Dithiothreitol (DTT), acrylamide, bis-acrylamide, urea, ammonium persulfate (APS) and Proteinase K were purchased from Sigma-Aldrich, United States. Bromophenol blue, Tetramethylethylenediamine (TEMED), and Ethylenediaminetetraacetic acid (EDTA) were procured from BIO-RAD, United States.
Phenylmethylsulfonyl fluoride
Manufactured by Avantor
Sourced in United States
Phenylmethylsulfonyl fluoride is a chemical compound commonly used as a protease inhibitor in biological research. It functions by irreversibly inhibiting serine proteases, which are enzymes that cleave peptide bonds. This product is primarily used in the preservation and stabilization of protein samples during extraction and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Bromophenol blue, by Bio-Rad (3 mentions)
Glycine, by Avantor (2 mentions)
Ethylenediaminetetraacetic acid, by Bio-Rad (2 mentions)
Bis acrylamide, by Merck Group (2 mentions)
Tetramethylethylenediamine, by Bio-Rad (2 mentions)
Tris buffer, by Avantor (2 mentions)
Low molecular weight marker for sds page, by GE Healthcare (2 mentions)
Yeast extract, by BD (2 mentions)
Urea, by Merck Group (2 mentions)
Tryptone, by BD (2 mentions)
Sodium dodecyl sulphate (sds), by Avantor (2 mentions)
Deae sepharose fast flow media, by GE Healthcare (2 mentions)
Protease inhibitor cocktail, by Roche (2 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
Sodium dodecyl sulfate (sds), by Avantor (1 mentions)
Isopropyl β d 1 thiogalactopyranoside (iptg), by Avantor (1 mentions)
Proteinase k, by Merck Group (1 mentions)
Ammonium persulfate, by Merck Group (1 mentions)
Dithiothreitol (dtt), by Merck Group (1 mentions)
Acrylamide, by Merck Group (1 mentions)
13 protocols using phenylmethylsulfonyl fluoride
1
Purification and Characterization of Proteins
2
Western Blot Analysis of Autophagy Markers
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Radioimmunoprecipitation assay (RIPA) buffer containing protease inhibitors (Roche, Indianapolis, USA) and 1 mM phenylmethylsulfonyl fluoride (Amresco, Solon, USA) was used to prepare cell lysates. Total proteins were separated on 12% SDS polyacrylamide gels followed by the staining with primary antibodies against LC3, beclin 1, mTOR, 4E-BP1, p85S6K, p70S6K, phospho-mTOR, phospho-4E-BP1, phospho-p85S6K, or phospho-p70S6K. GAPDH and β-actin were used for internal normalization. After the incubation with designated horseradish peroxidase-conjugated secondary antibodies, the bands were imaged using enhanced chemiluminescence (Amersham Bioscience, UK) and the band density was quantified using ImageJ software (NIH).
3
Western Blot Analysis of Histone Modifications
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Cell extracts were prepared in cold RIPA buffer (25 mmol/L Tris‐HCl, pH 7.6, 150 mmol/L NaCl, 1% Nonidet P‐40 (Sigma, 74 385), 1% sodium deoxycholate (Amresco, D0613), 0.1% sodium dodecyl sulphate (Amresco, 0227) and supplemented with 1 mmol/L phenylmethylsulfonyl fluoride (Amresco, M145) and a protein inhibitor cocktail (Roche Diagnostics, 04693132001). After transient sonication, the cell lysate was incubated on ice for 30 minutes. The samples were centrifuged at 12 000 rpm for 15 minutes to pellet the cell debris, and the supernatant was transferred to a new tube for further analysis. The protein lysates were separated by SDS‐PAGE and electro‐transferred onto a nitrocellulose membrane. The membrane was incubated with corresponding primary antibodies. Primary antibodies included anti‐RNF20 (Proteintech, 21625‐1‐AP, 1:1000), anti‐H2Bub (Cell Signaling Technology, 5546s, 1:1000), anti‐GAPDH (Bo Ao Rui Jing,ab1019t, 1:1000), anti‐Actin (Abmart, M20011, 1:1000), anti‐SOX2 (Santa Claus, sc365823X, 1:1000), anti‐H3 (Proteintech, 17168‐1‐AP, 1:1000) and anti‐H3K9me2 (EASYBIO, BE3283, 1:1000). The next day, the proteins on the membrane were hybridized with Alexa Fluor 680‐conjugated goat anti‐mouse or Alexa Fluor 800‐conjugated goat anti‐rabbit secondary antibodies and scanned using the ODYSSEY Sa Infrared Imaging System (LI‐COR biosciences).
4
Western Blot Analysis of Smo and Gli1
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Western blot assay of Smo and Gli1 proteins was performed at 7 days of the experiment. Brain tissue samples were dissolved in radio immunoprecipitation assay lysis buffer containing 1% phenylmethyl sulfonylfluoride (Amresco, Atlanta, GA, USA), and centrifuged at 13,000 × g for 10 minutes at 4°C. Samples (50 μg protein) were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). Membranes were blocked using 5% skimmed milk for 1 hour at room temperature, and then incubated with one of the following primary antibodies: rabbit polyclonal anti-Smo (1:1000; Abcam) or rabbit polyclonal anti-Gli1 (1:1000; Abcam) overnight at 4°C. Membranes were incubated in goat anti-rabbit secondary antibody (1:10,000; TDY Biotech, Beijing, China) for 1 hour at 37°C. Proteins bands were visualized using an enhanced chemiluminescence method (Xu et al., 2017). Images were captured and analyzed using an image analysis system (GelDoc XR+; ProteinSimple, San Jose, CA, USA). Glyceraldehyde-3-phosphate (GAPDH; Millipore, Billerica, MA, USA) was used as the internal control. Protein concentration was measured by bicinchoninic acid assay (Xu et al., 2017). The ratio of protein optical density ratio to internal control protein was calculated.
5
Purification and Characterization of L-Asparaginase
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Culture media components such as tryptone extract and Bacto yeast extract were purchased from Difco Laboratories, India. Phenylmethylsulfonyl fluoride (PMSF), isopropyl β-D -1-thiogalactopyranoside (IPTG), acrylamide, bis-acrylamide, Tris–HCl, and L -asparagine were from Amresco, United States. Sodium dodecyl sulfate (SDS), ammonium persulfate (APS), dithiothreitol (DTT), urea, Nessler’s reagent, and trichloroacetic acid (TCA) were from Sigma-Aldrich, United States. 2,2,2-Trifluoroethanol was from SRL, India. Tetramethyl ethylenediamine (TEMED), ethylenediaminetetraacetic acid (EDTA), and bromophenol blue were from Bio-Rad, United States. Coomassie Brilliant Blue R-250 and ampicillin were from USB Corporation, United States. Glacial acetic acid and methanol were from Merck’s EMPARTA®. Ethanol, n-propanol, and glycerol were of analytical grade and were from Spectrochem, India. DEAE-Sepharose Fast Flow media was purchased from GE Healthcare, United Kingdom. BCA assay kit, SDS-PAGE prestained molecular weight marker, and commercial r-hGH were from Thermo Fisher Scientific, United States. Commercial L -asparaginase 5,000 IU/ml, Bionase® 5K, was purchased from Zydus Cadila, India. RPMI, horse serum (HS), and fetal bovine serum (FBS) were from Gibco BRL, United States. All the other chemicals were of analytical grade.
6
Western Blot Analysis of Renal Proteins
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Renal tissues were homogenized in RIPA lysis buffer containing 1 mM phenylmethylsulfonyl fluoride (Amresco, Solon, OH, USA) and protease inhibitor cocktail (Roche, Indianapolis, IN, USA). Fifty micrograms of proteins was then separated by 12% SDS-PAGE and next electrophoretically transferred onto PVDF membranes. After blocking with 5% non-fat milk 1 h at room temperature, the membranes were probed with indicated primary antibodies at 4 °C overnight, followed by incubation with an HRP-conjugated secondary antibody. The reactive bands were visualized with ECL plus reagents as previously described.33 (link) The relative expression levels for a particular target were assessed by densitometric analysis and normalized by GADPH using the Image J software (http://rsb.info.nih.gov/ij/ ) as instructed.
7
Diabetic Nephropathy Pathogenic Mechanisms
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Dimethyl sulfoxide (Sigma-Aldrich, United States), high-sugar Dulbecco’s modified Eagle’s medium (HyClone, United States), low-sugar Dulbecco’s modified Eagle’s medium (HyClone), Cell Counting Kit-8 (CCK-8; Beyotime, China), fetal bovine serum (Amresco, United States), radioimmunoprecipitation assay buffer (Amresco), protein concentration detection kit (Amresco), phenylmethyl sulfonyl fluoride (Amresco), cocktail protease inhibitor (Amresco), sodium dodecyl sulfate (SDS; Amresco), STZ (MedChemExpress, United States), KN-93 (MedChemExpress), Tween-20 (Amresco), SAR (Sigma-Aldrich), primary antibodies against TRPC6 (Abcam, United States), CC3 (Abcam), T-CAMKII (Abcam), P-CAMKII (Signalway Antibody, United States), Bcl-2 (Abcam), glyceraldehyde 3-phosphate dehydrogenase (Cell Signaling Technology, United States), Bax (Cell Signaling Technology), PCNA (Abcam), CyclinD1 (Cell Signaling Technology), and sheep anti-mouse secondary antibody (Abcam) were used in this study.
8
Immunoblotting Analysis of Autophagy and Apoptosis Markers
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The U87MG and A172 cells were homogenized in radioimmunoprecipitation assay (RIPA) buffer [50 mM Tris–HCl (pH 7.4), 0.1% SDS, 1% Triton X-100, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, and 1 mM Na3VO4] containing 1 mM phenylmethylsulfonyl fluoride (Amresco, Solon, Ohio, USA) and protease inhibitor cocktail (Roche, Indianapolis, IN, USA). The protein samples were separated on a 12% SDS polyacrylamide gel and detected with antibodies against LC3, Beclin-1, Bax, Bcl-2, phospho-mTOR/mTOR, phospho-4E-BP1/4E-BP1, phospho-p85S6K/p85S6K, or phospho- p70S6K/p70S6K. β-actin (KC-5A08, Kangcheng, China) or GAPDH (KC-5G5, Kangcheng, China) were used as a loading control, and then the blots were probed with appropriate HRP-conjugated secondary antibodies. The protein bands were detected by enhanced chemiluminescence (ECL) (Amersham Bioscience, UK) and quantified using the Image J software program (NIH).
9
Protein Extraction and Western Blot Analysis
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Total protein was extracted using a cocktail of RIPA lysis buffer (VWR Life Science, cat# N653-100ML), protease inhibitor tablets (Thermo scientific, cat# A32953), and Phenyl methylsulfonyl fluoride (Amresco, cat# M145-5G). The protein concentration was determined with Bradford reagent (Biorad, cat# 500-0205). Primary antibodies used were against PCNA (2586S, Cell signaling Technology, Danvers, MA, USA) and α tubulin (SC32293, Santa Cruz Biotechnology, Dallas, TX, USA). Secondary antibodies used were either against mouse or rabbit (Sigma-Aldrich, St. Louis, MO, USA), as appropriate. Li-COR Odyssey CLx with infrared fluorescence, IRDye secondary antibodies and imagers were used to detect western blots without film or chemiluminescent substrates. All antibodies were made in 5% BSA. The primary antibodies were prepared using 1:1000 dilution and incubated overnight at 4 °C. For secondary antibodies, we used 1:10,000 dilution and incubated for 2 h at room temperature. Western blots were analyzed and quantified with Odyssey imager; Image Studio version 5. α tubulin was used as loading control.
10
Recombinant Protein Expression Protocol
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Components for culture media, tryptone and yeast extract were purchased from Difco Laboratories, India. Glycine, phenylmethylsulfonyl fluoride (PMSF), isopropyl β-d -1-thiogalactopyranoside (IPTG), Tris buffer and sodium dodecyl sulphate (SDS) were from Amresco, USA. Glucose and NaCl were purchased from Qualigen, India. Low molecular weight marker for SDS-PAGE and DEAE-Sepharose Fast Flow media were from GE Healthcare, USA. Dithiothreitol (DTT), acrylamide, bis-acrylamide, urea, ammonium persulphate (APS), TFE, NATA and ovalbumin were purchased from Sigma-Aldrich, USA. Bromophenol blue, Tetramethylethylenediamine (TEMED) and Ethylenediaminetetraacetic acid (EDTA) were procured from BIO-RAD, USA. Micro BCA assay kit was purchased from Pierce, USA.
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