Cells from 6-well culture dishes were scraped into 300 μl RIPA buffer (50 mM Tris pH 8.0, 150 mM NaCl, 1% NP-40, 5 mM EDTA, 0.5% sodium deoxycholate, 0.1% SDS) containing 1 mM phenylmethylsulfonyl fluoride (PMSF), a mixture of protease inhibitors (1 μM pepstatin, leupeptin, N-p-Tosyl-l-phenylalanine chloromethyl ketone, Nα-Tosyl-l-lysine chloromethyl ketone hydrochloride, trypsin inhibitor; Sigma), and a mixture of phosphatase inhibitors (2 mM imidazole, 1 mM NaF, 1 mM sodium orthovanadate; Sigma). Cell homogenates were sonicated and centrifuged at 100,000xg for 30 min at 4°C to provide a RIPA soluble fraction. Soluble lysates were analyzed by immunoblotting using the described antibodies. The antibodies used for western analysis were as follows: polyclonal anti-acetylated-lysine (1:1000, Cell Signaling #9441), anti-tau C-terminal clone T46 (1:1000, Thermofisher), and anti-acetylated tau, Ac-K280 [3 (link)] (1:1000), polyclonal anti-GFP (1:1000, Santa Cruz).
N p tosyl l phenylalanine chloromethyl ketone
Manufactured by Merck Group
Sourced in United States
N-p-Tosyl-l-phenylalanine chloromethyl ketone is a chemical compound used as a lab equipment product. It serves as a protease inhibitor, primarily targeting serine proteases.
Automatically generated - may contain errors
Lab products found in correlation
Nα tosyl l lysine chloromethyl ketone hydrochloride, by Merck Group (4 mentions)
Pepstatin, by Merck Group (3 mentions)
Trypsin inhibitor, by Merck Group (3 mentions)
2 mercaptoethanol, by Merck Group (3 mentions)
Benzamidine, by Merck Group (3 mentions)
Imidazole, by Merck Group (2 mentions)
Phenylmethanesulfonyl fluoride, by Merck Group (2 mentions)
Milli q system, by Merck Group (2 mentions)
Ammonium acetate, by Merck Group (2 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
Leupeptin, by Merck Group (1 mentions)
Adenosine 5 diphosphate, by Merck Group (1 mentions)
Oligomycin a, by Merck Group (1 mentions)
Glycerol, by Merck Group (1 mentions)
Dialysis tubing, by Thermo Fisher Scientific (1 mentions)
Glibenclamide, by Merck Group (1 mentions)
Glipizide, by Merck Group (1 mentions)
Glycerol, by AppliChem (1 mentions)
Ethylenediaminetetraacetic acid edta, by Scharlab (1 mentions)
Adenosine 5 triphosphate, by Merck Group (1 mentions)
7 protocols using n p tosyl l phenylalanine chloromethyl ketone
1
Immunoblotting Analysis of Acetylated Proteins
2
Biochemical Reagents for Protein Analysis
3
Comprehensive Analytical Reagent Protocol
4
HEK-293A Plasmid Transfection and Protein Extraction
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Two micrograms of desired DNA plasmid were transfected into HEK-293A cells with Fugene-6 (Promega) at 2.5 μL Fugene per 1 μg DNA plasmid ratio. Twenty-four hours post transfection 5 mM MG132 was added per well overnight. Forty-eight hours post transfection cells were harvested in 250 μL 1X RIPA buffer (50 mM Tris pH 8.0, 150 mM NaCl, 1% NP-40, 5 mM EDTA, 0.5% sodium deoxycholate, 0.1% SDS) with protease, phosphatase and deacetylase inhibitors (1 mg/mL pepstatin, leuptin, N-p-Tosyl-L-phenylalanine chloromethyl ketone, Nα-Tosyl-L-lysine chloromethyl ketone hydrochloride, trypsin inhibitor; Sigma, 1 mM NaF, 1 mM sodium orthovanadate, and 1 mM beta-glycerophosphate; Sigma, 2 μM TSA, 10 mM NCA). Samples were sonicated 20 times and then centrifuged at 21,130g for 30 min at 4°C. Supernatant (soluble fraction) was collected and analyzed by immunoblotting. Reaction was finished by adding loading dye solution, boiled for 10 min at 98°C and analyzed by immunoblotting.
5
Fractionation and Analysis of Tau Protein
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Fractionation of all cell lysates was performed by sequential extraction using buffers of increasing strength. Cells from 6-well culture dishes were scraped into 300 μl RIPA buffer (50 mM Tris pH 8.0, 150 mM NaCl, 1% NP-40, 5 mM EDTA, 0.5% sodium deoxycholate, 0.1% SDS) containing 1 mM phenylmethylsulfonyl fluoride (PMSF), a mixture of protease inhibitors (1 μM pepstatin, leupeptin, N-p-Tosyl-l-phenylalanine chloromethyl ketone, Nα-Tosyl-l-lysine chloromethyl ketone hydrochloride, trypsin inhibitor; Sigma), and a mixture of phosphatase inhibitors (2 mM imidazole, 1 mM NaF, 1 mM sodium orthovanadate; Sigma). Samples were sonicated and centrifuged at 100,000xg for 30 min at 4°C, and then re-extracted in RIPA buffer to ensure complete removal of soluble proteins. Resultant insoluble pellets were extracted in 100 μl urea buffer (7 M urea, 2 M Thiourea, 4% CHAPS, 30 mM Tris, pH 8.5) containing the same formulation of inhibitors as described for RIPA buffer, then sonicated and centrifuged at 100,000xg for 30 min at room temperature. Soluble and insoluble fractions were analyzed by western blotting using the indicated antibodies. Tau antibodies used for western analysis were as follows: C-terminal T46 (1:1000, Thermofisher), tau-5 (1:1000, Thermofisher), tau-1 (1:1000, Thermofisher), K9JA (1:10,000, Dako), ac-K280 [3 (link)] (1:1000), and ac-K163 (1:1000, this study).
6
Protein Isolation and Purification
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Dimethyl sulfoxide (DMSO), ethylenediaminetetraacetic acid (EDTA), Bis-Tris, glycine, sodium dodecyl sulphate (SDS), dithiothreitol (DTT), benzamidine, 2-mercaptoethanol, phenyl methyl sulphonyl fluoride (PMSF), soybean trypsin inhibitor, tosyl-lysyl-chloromethyl ketone (TLCK), N-p-Tosyl-l -phenylalanine chloromethyl ketone (TPCK), 2-Amino-2-(hydroxymethyl)-1,3-propanediol (TRIS) and bovine albumin protein standard were from Sigma Aldrich, St. Louis, MO, USA. Calcium chloride dihydrate, sodium chloride, sodium hydroxide, isopropanol and hydrochloric acid were from Merck, Darmstadt, Germany. Carboxybenzyl-Gly-Pro-Arg p-nitroanilide acetate salt (CBZ-GPR-pNA) was from Bachem AG, Bubendorf, Switzerland. ECL Plex goat Anti-rabbit IgG Cy5, ECL Plex goat Anti-mouse IgG Cy3 were from GE Healthcare, Chalfont St. Giles, UK. Micro BCA Protein Assay Kit was from Thermo Scientific. benzamidine purified cod trypsin was from Zymetech (Reykjavik, Iceland).
7
Inhibitor Compounds for Cell Studies
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N-p-tosyl-L-phenylalanine chloromethyl ketone (TPCK, # T4376), N-α-tosyl-L-lysine chloromethyl ketone hydrochloride (TLCK, #90182), suberoylanilide hydroxamic acid (SAHA, #SML0061), anacardic acid (#A7236), decitabine (#A3656), lipopolysaccharide (LPS from Escherichia coli O111:B4, #L4391) were purchased from Sigma-Aldrich. Carfilzomib (#S2853) and tocilizumab (#A2012) were purchased from Selleckchem. Ro-106-9920 (#1178), TPCA-1 (#2559), PS1145 (#4569), Bay11-7082 (#1744), Ruxolinitib (#7048) and Stattic (#2798) were purchased from TOCRIS bioscience. TAK-242 (# 614316) was purchased from Millipore. Etanercept was supplied by Pfizer.
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