Tissue and cell lysates were extracted using RIPA lysis buffer with the protease inhibitor phenylmethane sulfonyl fluoride (Merck Millipore, USA). Protein concentration was measured using the BCA protein assay kit (Beyotime Biotechnology) according to the manufacturer's instructions. Equivalent amounts of protein (30μg) were separated on 10% SDS PAGE gel and then transferred onto 0.45μm PVDF membranes (Millipore, Billeria, MA) according to the standard protocols. Membranes were blocked in 5% milk in TBST buffer for 1 hr at room temperature, followed by incubation with primary antibodies at 4°C overnight. After incubation with a secondary antibody for 1 hr at room temperature, proteins were detected using ECL regent (Millipore, Billeria, MA). Primary antibodies were as follows: Barx1 (Santa Cruz, sc-81956), β-actin (sc-47778), MGAT5 (Abcam, ab87977), and MMP9 (Cell Signaling, #13667).
Phenylmethanesulfonyl fluoride
Manufactured by Merck Group
Sourced in United States, Germany, China, Macao, Canada
Phenylmethanesulfonyl fluoride is a laboratory reagent used as a serine protease inhibitor. It functions by irreversibly inhibiting the activity of serine proteases, which are enzymes that play a role in various biological processes. This compound is commonly used in biochemical research and applications where controlling serine protease activity is important.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor cocktail, by Merck Group (35 mentions)
Leupeptin, by Merck Group (17 mentions)
Triton x 100, by Merck Group (15 mentions)
Aprotinin, by Merck Group (15 mentions)
Protease inhibitor, by Merck Group (15 mentions)
Bovine serum albumin (bsa), by Merck Group (15 mentions)
Phosphatase inhibitor, by Merck Group (14 mentions)
Dithiothreitol (dtt), by Merck Group (14 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (14 mentions)
Sodium dodecyl sulfate (sds), by Merck Group (13 mentions)
Sodium orthovanadate, by Merck Group (11 mentions)
Precision plus protein kaleidoscope, by Bio-Rad (10 mentions)
Cell lysis buffer, by Cell Signaling Technology (10 mentions)
Glycerol, by Merck Group (9 mentions)
Tween 20, by Merck Group (9 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (9 mentions)
Sodium chloride (nacl), by Merck Group (9 mentions)
Stripping solution, by Bio-Rad (7 mentions)
Luminata classico, by Merck Group (7 mentions)
Methanol, by Merck Group (7 mentions)
241 protocols using phenylmethanesulfonyl fluoride
1
Protein Expression Analysis Protocol
2
Cytokine Profiling in Colitis and CRC
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
IL-1β (cat. no. KT2040-A), IL-2 (cat. no. KT2698-A), IL-6 (cat. no. KT2163-A), IL-8 (cat. no. KT2123-A), IL-15 (cat. no. KT2172-A), IL-17 (cat. no. KT2170-A), IL-22 (cat. no. KT9441-A), TNF-α (cat. no. KT2132-A), IFN-γ (cat. no. KT2182-A) concentrations in the colon tissues, which were lysed using Radio Immunoprecipitation Assay (cat. no. R0010; Beijing Solarbio Science & Technology Co., Ltd.) containing 1% protease inhibitor cocktail (cat. no. P8340; Sigma-Aldrich; Merck KGaA) and 2% phenylmethanesulfonyl fluoride (cat. no. P7626; Sigma-Aldrich; Merck KGaA), and/or serum of mice with UC and CRC were measured using commercialized ELISA kits (Jiangsu Kete Bio-Technology Co., Ltd.) according to the manufacturer's protocols.
3
Vinblastine-Induced Protein Dephosphorylation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
When cells treated with vinblastine were harvested, the pellets were separated in two: one where total proteins were extracted in the presence of phosphatase inhibitors (10 mM NaF, 1 mM Na3VO4, 1 mM phenylmethanesulfonylfluoride, Sigma-Aldrich), and one where phosphatase inhibitors were omitted. The latter extracts were submitted to dephosphorylation with 200 units of λ-phosphatase (#P0753S, Biolabs, Ipswich, MA, USA) for 2 h at 37 °C. The reaction was stopped by 4% SDS, 125 mM tris pH = 6.8, 20% glycerol (Sigma-Aldrich), 0.002% (w/v) bromophenol blue (Sigma-Aldrich).
4
Reagents and Oligonucleotides for Telomere Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Potassium
hexacyanoferrate(III) (99.98%),
potassium hexacyanoferrate(II) trihydrate (≥99.95%), ethylenediaminetetraacetic
acid (EDTA) ACS reagent (99.4–100.6%), 2-propanol anhydrous
(99.5%), sodium chloride (≥99.95%), acetic acid (≥99%),
Tween 20, Trizma base (99.9%) Trizma hydrochloride (≥99%),
methanol (≥99%), phenylmethanesulfonyl fluoride (PMSF), diethyl
pyrocarbonate (DCEP) (≥97%),dl -dithiothreitol (DTT)
(≥98%), glycine (≥99%), sodium dodecyl sulfate (SDS;
≥98.5%), and ddGTPs were purchased from Sigma-Aldrich (St.
Louis, MO). Acrylamide (40%), ammonium persulfate, tetramethylethylenediamine,
4× Laemmli sample buffer, and precision plus protein standard
were obtained from BioRad (Hercules, CA). Tris(2-carboxyethyl) phosphine
hydrochloride (TCEP), sulfuric acid optima (93–98%), Coomassie
Brilliant Blue G-250, HEPES buffer, detergent-compatible Bradford
assay kit, and RPMI-164 medium + 2.05 mM glutamine (Hyclone) were
purchased from Fisher Scientific (Fair-lawn, NJ). All synthetic oligonucleotides
were purchased from Integrated DNA Technology (IDT) (San Diego, CA)
with the following sequences:
TS30 (5′-S-S-(CH2)6-TTTTTTTTTTAATCCGTCGAGCAGAGTT-3′),
TS60 (5′-S-S-(CH2)6-TTTTTTTTTTAATCCGTCGAGCAGAGTTAGGGTTAGGGTTAGGGTTAGGGTTAGG-3′),
and
TSC (5′-S-S-(CH2)6-AAAAAAAAAATTAGGCAGCTCGTCTCAA-3′).
dNTPs were purchased
from Promega (Madison, WI, USA).
hexacyanoferrate(III) (99.98%),
potassium hexacyanoferrate(II) trihydrate (≥99.95%), ethylenediaminetetraacetic
acid (EDTA) ACS reagent (99.4–100.6%), 2-propanol anhydrous
(99.5%), sodium chloride (≥99.95%), acetic acid (≥99%),
Tween 20, Trizma base (99.9%) Trizma hydrochloride (≥99%),
methanol (≥99%), phenylmethanesulfonyl fluoride (PMSF), diethyl
pyrocarbonate (DCEP) (≥97%),
(≥98%), glycine (≥99%), sodium dodecyl sulfate (SDS;
≥98.5%), and ddGTPs were purchased from Sigma-Aldrich (St.
Louis, MO). Acrylamide (40%), ammonium persulfate, tetramethylethylenediamine,
4× Laemmli sample buffer, and precision plus protein standard
were obtained from BioRad (Hercules, CA). Tris(2-carboxyethyl) phosphine
hydrochloride (TCEP), sulfuric acid optima (93–98%), Coomassie
Brilliant Blue G-250, HEPES buffer, detergent-compatible Bradford
assay kit, and RPMI-164 medium + 2.05 mM glutamine (Hyclone) were
purchased from Fisher Scientific (Fair-lawn, NJ). All synthetic oligonucleotides
were purchased from Integrated DNA Technology (IDT) (San Diego, CA)
with the following sequences:
and
dNTPs were purchased
from Promega (Madison, WI, USA).
5
Western Blot Analysis of Cellular Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For the preparation of cellular lysates, cells were harvested with immunoprecipitation assay buffer (Beyotime) supplemented with a proteinase inhibitor cocktail (Roche, USA) and phenylmethanesulfonyl fluoride (Sigma-Aldrich). Protein concentration was determined using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, USA). Protein samples were heat denatured and separated by sodium dodecyl sulfate (SDS)-PAGE gel-electrophoresis and transferred to PVDF membranes (Millipore, USA). After blocking in phosphate-buffered saline (PBS)/Tween-20 containing 5% nonfat milk, the membranes were incubated with primary antibodies overnight at 4 °C, followed by incubation with HRP-conjugated anti-rabbit/mouse secondary IgG antibodies (CST, USA) for an hour. Then the protein bands were visualized using SuperSignal West Pico PLUS (Thermo Fisher Scientific) and analyzed by ImageQuant LAS 4000 mini (GE-Healthcare, USA). The information for primary antibodies is presented in Additional file 4 . Original blots can be found in Additional file 5 .
6
Characterization of Multidrug Resistance Transporters
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
R6G, KTC, ANI, MIC, CTR, CYH, ITC, NR, adenosine triphosphate (ATP), oligomycin (OM), trypsin, phenylmethanesulfonyl fluoride (PMSF), p-tosyl-L-lysine chloromethyl ketone (TLCK), and tosyl phenylalanyl chloromethyl ketone (TPCK) were procured from Sigma Chemical Co. (St. Louis, MO). The protease inhibitors leupeptin, pepstatin A, and aprotinin were purchased from G-Biosciences (MO, USA). FLC was generously provided by Ranbaxy, India. Ascorbic acid (AA) was purchased from SRL (Mumbai, India). Oligonucleotides used in this study were commercially procured from Sigma Genosys, India and are listed in Supplementary Table S4 . Anti-GFP monoclonal antibody and anti-mouse secondary antibody were purchased from Santa Cruz Biotechnology Inc. (Texas, USA). The anti-Pma1 (PM ATPase) polyclonal antibody was a gift from Professor Ramon Serrano (Universidad Politecnica de Valencia-CSIC, Valencia, Spain). 3H-radiolabeled FLC (3H-FLC) was purchased from Moravek Biochemicals and Radiochemicals (CA, USA).
7
Western Blot Analysis of BCL11A and LRF
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blot analysis was performed as described earlier27 (link). Briefly, whole-cell lysates were prepared using radioimmunoprecipitation assay buffer containing phenylmethanesulfonylfluoride (Sigma-Aldrich) and Halt Protease Inhibitor Cocktail (Thermo Scientific, Rockford, IL, USA). The lysate (30 μg) was loaded on a 7% sodium dodecyl sulfate-polyacrylamide gel and the western blotting was performed using the primary antibodies, anti-BCL11A (1:1000 dilution) (Cell Signaling Technologies, Danvers, MA, USA), anti-LRF (1:1000 dilution) (Cell Signaling Technologies) and anti-actin (1:5000 dilution) (BD Pharmingen), and the secondary antibodies, anti-mouse IgG HRP (Cell Signaling Technologies) and anti-rabbit IgG HRP (Invitrogen Corporation, Camarillo, CA, USA).
8
Biochemical Reagents for Protein Analysis
9
Evaluating Cytotoxicity in HBVSMCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The cytotoxicity in HBVSMCs was estimated by the mitochondrial-dependent reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT; Sigma-Aldrich) to purple formazan. Briefly, after treatments, cells were incubated with 0.5 mg/ml MTT solution for 90 min at 37 °C. The medium was replaced with dimethyl sulfoxide (Sigma-Aldrich) to dissolve formazan crystals, and the amount of formazan formed was spectrophotometrically quantified at 560 nm and 620 nm using a Synergy™ Mx Microplate reader (BioTek Instruments Inc.). Cell viability is expressed as a percentage of the control condition for each independent experiment.
Total cell lysates for western blot analysis were obtained by cell collection in freshly prepared ice-cold lysis buffer containing 50 mM Tris–HCl, 150 mM NaCl, 5 mM CaCl2, 0.05% BRIJ-35, 0.02% NaN3, 1% Triton X-100, 1% phenylmethanesulfonyl fluoride (Sigma-Aldrich), and 0.5% aprotinin (Sigma-Aldrich). The lysates were sonicated for 10 s and stored at -80 °C until use.
Total cell lysates for western blot analysis were obtained by cell collection in freshly prepared ice-cold lysis buffer containing 50 mM Tris–HCl, 150 mM NaCl, 5 mM CaCl2, 0.05% BRIJ-35, 0.02% NaN3, 1% Triton X-100, 1% phenylmethanesulfonyl fluoride (Sigma-Aldrich), and 0.5% aprotinin (Sigma-Aldrich). The lysates were sonicated for 10 s and stored at -80 °C until use.
10
Reagents and Chemicals for Cell Culture Studies
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Fetal Bovine Serum (FBS) and Phosphate Buffered Saline (PBS) were purchased from Hyclone™, ThermoFisher Scientific, Waltham, MA, USA. HEPES, EDTA, EGTA, TEMED, isopropanol, aprotonin, pepstatin, phenylmethanesulfonyl fluoride (PMSF), leupeptin, sodium fluoride (NaF), sodium orthovanadate (Na3VO4), diethyldithiocarbamate (DDC), doxorubicin, etoposide, fludarabine, 5-flouroUracil (5-FU), cisplatin, dimethyl-9,9’-biacridinium dinitrate (Lucigenin), N-acetyl-cysteine (NAC) were purchased from Sigma-Aldrich, St. Louis, MO, USA. Venetoclax (VEN) was purchased from Medchem Express LLC. Streptomycin–penicillin and l -glutamine were purchased from Gibco. Coomassie Blue dye and bovine serum albumins (BSA) were purchased from ThermoFisher Scientific. Methanol and sodium dodecyl sulphate (SDS) were purchased from Merck, Kenilworth, NJ, USA. Sucrose and 30% acrylamide/bis solution were purchased from Bio-Rad. Goat anti-mouse and anti-rabbit IgG horseradish peroxidase conjugated secondary antibodies were purchased from Pierce, TX, USA. Protein A agarose beads were purchased from Santa Cruz, TX, USA. Chloromethyl-2-,7-dichlorofluorescin diacetate (DCFDA) and MitoSox™ were purchased from Molecular Probes, Thermo Fisher Scientific.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!