Total protein extracts were obtained from 106 amoebae incubated in lysis buffer (10% SDS, 10 mM Tris/HCl) containing a protease inhibitor cocktail (20 mM leupeptine (Sigma-Aldrich, USA), 50 mM N-ethylmaleimide (Sigma-Aldrich, USA), 5 mM p-chloromercuribenzoate (Sigma-Aldrich, USA), 2 mM 4-(2-aminoethyl) benzenesulfonyl fluoride (Sigma-Aldrich, USA), 2 × complete mini EDTA-free (protease inhibitor cocktail, Roche, Suisse), a tablet of phosSTOP (Roche, Suisse), 10 mM E-64, 2 mM Na3VO4, 100 mM NaF, 10 mM iodoacetamide and 1% SDS). Crude cell extracts were boiled for 5 min at 100°C, cooled in ice for 1 min, aliquoted, and stored at −20°C. Protein samples (equivalent to 80,000 cells per lane) were resolved by 10% SDS-PAGE and electrotransferred onto a 0.2 μm PVDF membrane (Immobilon PSQ, Millipore). Proteins were detected by immunoblotting using mouse anti-actin monoclonal antibody (1:50 dilution; Clone C4, Merck Millipore, Germany), anti-HaloTag mouse monoclonal antibody (1:200 dilution, Promega, USA) or polyclonal anti-Arp3 (1:500 dilution, this work); the secondary antibodies were sheep peroxidase-conjugated anti-mouse (1:10,000; G&E) or anti-rabbit (1:20,000; G&E, USA) IgG. Membranes were treated with ECL Western blotting detection reagent and then exposed to Kodak Biomax film.
4 2 aminoethyl benzenesulfonyl fluoride
Manufactured by Merck Group
Sourced in United States
4-(2-aminoethyl) benzenesulfonyl fluoride is a chemical compound that is commonly used as a lab equipment product. It serves as a core function in various research and analytical applications.
Automatically generated - may contain errors
Lab products found in correlation
N ethylmaleimide, by Merck Group (1 mentions)
Immobilon psq, by Merck Group (1 mentions)
Clone c4, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Mouse anti actin, by Merck Group (1 mentions)
Leupeptine, by Merck Group (1 mentions)
Safire2 microplate reader, by Tecan (1 mentions)
Proteinase k, by Merck Group (1 mentions)
Pepstatin a, by Merck Group (1 mentions)
Las 4000 imager, by Fujifilm (1 mentions)
Phosphatase inhibitor, by Merck Group (1 mentions)
Leupeptin, by Merck Group (1 mentions)
Aprotinin, by Merck Group (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Merck Group (1 mentions)
Whatman, by Cytiva (1 mentions)
Las 4000, by Cytiva (1 mentions)
Sodium orthovanadate, by Merck Group (1 mentions)
Human recombinant ptn, by Thermo Fisher Scientific (1 mentions)
Allopurinol, by Merck Group (1 mentions)
U0126, by Bio-Techne (1 mentions)
5 protocols using 4 2 aminoethyl benzenesulfonyl fluoride
1
Amoeba Protein Extraction and Western Blot Analysis
2
Evaluating Exosomal Aβ Localization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To examine whether exosome-associated Aβ is luminal or bound to the exterior exosome surface, exosomes were isolated from conditioned media (oAβAF700-treated) of dSH-SY5Y cells and incubated with proteinase K (Sigma-Aldrich,1 mg/ml) for 30 min at 37 °C. 4-(2-aminoethyl)-benzene-sulfonyl fluoride (Sigma-Aldrich, 0.5 mM) was subsequently added to the vesicle fraction to inactivate the enzyme prior to two rounds of 100,000 × g centrifugation. The final pellet was resuspended in PBS and AF700 fluorescence was measured in Tecan Safire2 microplate reader at Ex/Em 696/719 nm.
3
Protein Extraction and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Whole cell lysates were prepared by extracting proteins using a cold radioimmunoprecipitation assay buffer [50 mM Tris-HCl, pH 7.4; 150 mM NaCl; 1% Nonidet P-40; and 0.1% sodium dodecylsulfate (SDS); supplemented with protease inhibitors (10 μg/ml leupeptin, 10 μg/ml pepstatin A, 10 μg/ml aprotinin and 1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride and phosphatase inhibitors (1 mM NaF and 1 mM Na3VO4)] obtained from Sigma-Aldrich. The lysates were size-fractionated by SDS-polyacrylamide gel electrophoresis and transferred to a nitrocellulose (NC) membrane (Whatman Schleicher and Schuell, Dassel, Germany). The NC sheet was then blocked with 5% non-fat dry milk in Tris-buffered saline. Antibodies against type II collagen, COX-2, pp38, pERK, pJNK and Akt were used for probing corresponding NC blots overnight at 4°C. The membranes were then washed three times with Tris-buffered saline/Tween-20 and incubated with horseradish peroxidase-conjugated secondary antibody (Sigma-Aldrich) for 2 h followed by exposure in an LAS-4000 imager (Fujifilm Corp., Tokyo, Japan) according to the manufacturer’s instructions. The experimental results were transformed into in numerical values using Image J 1.41 (Software Inquiry, Quebec, Canada).
4
Fecal Sample Collection and Processing
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Two thumb-sized fresh stool samples each weighing approximately 0.2–0.4 g (HPS1 and CS1) were collected from each participant in the morning. A second stool sample (HPS2) was collected from the H. pylori-infected group 4 weeks after yogurt ingestion. Third fecal samples (HPS3 and CS3) were collected at the completion of the 4 weeks of treatment and/or yogurt ingestion in both groups. Total fecal DNA was extracted using a Qiagen stool kit (Qiagen, Chatworth, CA, USA) and then stored as −20 °C. The other sample was diluted in PBS containing 2.5 mg/mL leupeptin, 11 mg/mL aprotinin, and 0.5 mM 4-(2-aminoethyl) benzenesulfonyl fluoride (Sigma, St. Louis, MO, USA). After thorough mixing and centrifugation for 10 min at 10,000 g, the supernatant was stored as −80 °C for further ELISA tests.
5
Antioxidant and Kinase Inhibitor Evaluation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Human recombinant PTN was from PeproTech, Inc. (Rocky Hill, NJ, USA). The NAD(P)H oxidase-specific inhibitor 4-hydroxy-3methoxyacetophenone (apocynin) and the ROS-sensitive fluorescent dye, 5(6)-carboxy-2,7-dichlorodihydrofluorescein diacetate (carboxy-H 2 DCFDA) were purchased from Fluka. The hydrogen peroxide scavenger catalase, the NAD(P)H oxidase inhibitors 4-(2aminoethyl)benzenesulfonylfluoride (AEBSF) and 3-benzyl-7-(2benzoxazolyl)thio-1,2,3-triazolo(4,5-d)pyrimidine (VAS2780), the XO inhibitor allopurinol and the general phosphatase inhibitor sodium orthovanadate were purchased from Sigma. The XO inhibitor febuxostat was from Santa Cruz Biotechnology, Inc. The mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK) inhibitor U0126 and the src kinase inhibitor PP1 were purchased from Tocris (Ellisville, MO, USA). RNA oligonucleotide primers for RPTPβ/ζ (Polykratis et al., 2005) were obtained from VBCBiotech Services (Vienna, Austria), and for human XO were obtained from Santa Cruz Biotechnology, Inc. Double-stranded negative control siRNA was from Ambion (Austin, TX, USA). B3 (CYDMKTTC) and B3 scrambled (TKCMTCDY) peptides were from Cambridge Peptides (Birmingham, UK). All the inhibitors and antioxidants at the concentrations used were not toxic to the cells (data not shown).
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!