PMSF

Phenylmethanesulfonyl fluoride (PMSF) [329-98-6] M 174.2, m 90-91 , 92-93 . Purified by recrystn from ""CgHe, pet ether or CHCl3-pet ether. [Davies and Dick J Chem Soc 483 1932 cf Tullock and Coffman J Org Chem 23 2016 I 960.] It is a general protease inhibitor (specific for trypsin and chymotrypsin) and is a good substitute for diisopropylphosphoro floridate [Fahrney and Gould 7 Am Chem Soc 85 997 1963]. 

Inhibitors which interact only with peptidases of one catalytic type include pepstatin (aspartic peptidases) E64 (cysteine peptidases from clan CA) diisopropyl fluorophosphates (DFP) and phenylmethane sulfonyl-fluoride (PMSF) (serine peptidases). Bestatin is a useful inhibitor of aminopeptidases. 

The first compound to find widespread use as an inhibitor of AEA breakdown was phenylmethylsulfone (PMSF) (25). This non-selective serine protease inhibitor has been found to act as an irreversible inhibitor of FAAH preventing AEA breakdown [41, 47], and is commonly added to binding... 

Lysis buffer 400 mM KOAc, 25 mM KHEPES, pH 7.2, 15 mM Mg(OAc)2, 1% (v/v) NP-40, 0.5% (w/v) DOC, 1 mM DTT, ImM PMSF, 0.2 mM cycloheximide, 40 U/ml RNase Out. NP40 must be added before DOC to prevent DOC from precipitating. Long-term storage of this buffer containing detergent is not recommended. DTT, PMSF, cycloheximide, and RNase Out must be added fresh. 

From Sigma 3-aminoethylcarbazole (AEC) acrylamide/bis-acrylamide (30%) 37.5 1 amino acids alumina bentonite benzamidine bovine fiver tRNA bovine serum albumin (BSA) creatine phosphate (CP) diethyl pyrocarbonate (DEPC) dithiothreitol (DTT) Escherichia coli MRE600 tRNA pyrophosphatase (Ppase) Ca++ salt of folinic acid, (5-formyl THF) IIHPHS K salt of phospho-enol pyruvic acid, (PEP) creatine phospho kinase (CPK) protease inhibitor cocktail for fungal and yeast extracts phenylmethylsulfonyl fluoride (PMSF) spermidine trihydrochloride Tween 20. 

Buffer and chemicals pH 7.8 phosphate buffer (16.29g Na2HP04-2H20, 1.17g NaH2P04H20, 1000 ml distilled water), Ethylene Diamine Tetra Acetic Acid (EDTA), Methionine (Met), Polyvinylpyrrolidone (PVP), Triton X-100, Phenylmethylsulphonylfluoride (PMSF), Riboflavin, Nitro Blue Tetrazolium (NBT). 

Recover the cells by centrifugation, lyse them in wash buffer containing protease inhibitors (e.g., 20 pM PMSF), and clarify the supernatant by centrifugation. 

BHT, potassium metabisulfite, PMSF and DTT are added just before homogenization, as these reagents readily inactivate. 

Thaw roots for 2 min by dipping into 50% dimethyl sulfoxide containing 0.1 MNa-cacodylate, pH 7.4,2 mM CaCl2,5 mMMgCl2, and 1 mM phenylmethyl-sulfonyl fluoride (PMSF). 

Samples are ready for treatment, if any. In this case, samples were washed in a buffer solution of 0.1 MNa-cacodylate, pH 7.4,4% sucrose, 1 mM PMSF and various concentrations of divalent cations. Incubation is for 10 min at 4°C with three rapid changes. 

During the protein isolation step, the presence of protease inhibitors is crucial to preserve the integrity of the antigen under study. Some protease inhibitors, such as phenybnethylsulfonylfluoride (PMSF), have a wide spectrum of activity and are therefore commonly used, whereas other inhibitors, such as okadaic acid are used primarily when the phosphorylated form of protein is of interest. O Table 8-3 contains a list of some commonly used protease inhibitors and their activity spectra. 

Three preliminary experiments were carried out the first done as described above, the second utilized a modified buffer containing protectants, and the third utilized both the modified buffer and a hemoglobin gel step to remove proteases. The modified buffer had the following recipe 50 mM EPPS-KOH buffer at pH 8.6, 20% (v/v) glycerol, 1.0 mM EDTA, 1.0 mM DTT, 0.5 mM phenylmethylsulfonyl fluoride (PMSF), 0.05 mM leupeptin, and 0.05% / -mercaptoethanol. 

Figure 2. Zymogram of gCenA (A-I) and ngCenA (J-Q) after incubation with C. fimi protease. Cellulases, bound to Avicel, were incubated with protease or control buffer for 72 hr at 30° C, then centrifuged to give cellulose-bound (A-E, J-N) and supernatant (F-I, O-Q) fractions. Products were separated on a SDS gel, replicated onto CMC-agarose and developed with Congo red. A,J. buffer control (4°C incubation) B,F,K,0, protease C,G,L,P, protease + PMSF control D,H,M,Q, buffer control E,I,N, buffer + PMSF control.

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