An oligopeptide that is known to inhibit chymotrypsin-like proteases (for example, cathepsin A, B, and D, and papain).
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However, the inhibition of proteases using TPCK has been inefficient in other crustacean species (Garcia-Carreno & Haard, 1993; Garcia-Carreno et al., 1994; Fernandez-Gimenez et al., 2002; Perera et al., 2008; Buarque et al., 2010), suggesting that the use of other specific chymotrypsin inhibitors (e.g., chymostatin) should be further evaluated to corroborate the findings obtained in this research.
NTS samples were homogenized in an ice-cold lysing buffer containing 20 mM Tris-HCl (pH 6.8), 150 mM NaCl, 10% glycerol, 1% NP-40, and 8 [micro]L/mL inhibitor cocktail (125 mM PMSF, 2.5 mg/mL aprotinin, 2.5 mg/ mL leupeptin, 2.5 mg/mL antipain, and 2.5 mg/mL chymostatin).
For protein extraction, the tissue was homogenized at 4[degrees]C in Buffr C (50 mmol/L Tris-Cl, pH 7.5) containing 2 mmol/L DTT, 2 mmol/L EGTA, 2 mmol/L EDTA, 50 mmol/L 4-[2-aminoethyl]-benzenesulfonylfloride hydrochloride; 5 mg/ml each of leupeptin, aprotinin, pepstatin A, and chymostatin; and 50 mmol/L KF, 5 mmol/L sodium pyrophosphate, 50 mmol/L okadaic acid, and 2% SDS, and sonicated to dissolve the retinal tissue completely.
Western blotting was performed by lysing the cells in 20 mM Tris-HCl buffer (pH 7.4) containing a protease inhibitor mixture (0.1 mM phenylmethanesulfonyl fluoride, 5mg/mL aprotinin, 5mg/mL pepstatin A, and 1mg/mL chymostatin).
Chymostatin (0.5 mM well-1) was used as a positive control.(Table 7).
Frozen jejunal tissue mucosa samples (100 mg) were homogenized in 50 mM HEPES buffer (pH 7.4) containing 1 mM EDTA, 1 mM dithiothreitol, 5 mg/L phenylmethylsulfonyl fluoride, 5 mg/L aprotinin, 5 mg/L chymostatin, and 5 mg/L pepstatin added phosphatase inhibitor, sodium orthovanadate to a final concentration of 2 mM.
Effect of protease inhibitors on endogenous or exogenous [beta]-galactosidase in human fibroblasts Inhibitor Protease [beta]- [beta]- Galactosidase Galactosidase (endogenous) * (exogenous) ** None 100 100 E-64 Thiol 430 229 (Cysteine) *** Leupeptin Thiol 460 222 (Cysteine) *** Antipain Thiol 490 224 (Cysteine) *** Chymostatin Thiol 520 188 (Cysteine) *** Elastatinal Elastase 60 103 Pepstatin Pepsin 100 97 Phosphoramidon Metallopeptidase 110 88 Bestatin Aminopeptidase 110 90 * Relative enzyme activity in human fibroblasts with cathepsin A deficiency (galactosialidosis).
Cells were lysed with 20 mM Tris-HCl buffer (pH 7.4) containing protease inhibitors (0.1 mM phenylmethanesulfonyl fluoride, 5 mg/mL aprotinin, 5 mg/mL pepstatin A, and 1 mg/mL chymostatin).
8-bromoguanosine 3',5-icyclic monophosphate (8-Br-cGMP), Phenylmethylsulfonyl fluoride, orthovanadate, chymostatin, leuptin, antipain, pepstatin A, trypan blue, Tween-20, mouse anti-[alpha]-tubulin, 2-Propanol, chloroform, and TRI Reagent were purchased from Sigma Chemical (St Louis, MO, USA).
The cells lysates were prepared using an ice-cold lysis buffer (50 mM Tris, 150 mM NaCl, 10 mM EDTA, 1% Triton) supplemented with a mixture of protease inhibitors containing antipain, bestatin, chymostatin, leupeptin, pepstatin, phosphoramidon, pefabloc, EDTA, and aprotinin (Boehringer, Mannheim, Germany).
Lysis was performed by the addition of 50 [micro]L ice-cold, modified RIPA lysis buffer (50 mM Tris buffer, pH 7.4 containing 150 mM sodium chloride, 2% (v/v) NP40, 0.25% (w/v) sodium deoxycholate, 1 mM EGTA, 10 mM sodium orthovanadate, 0.5 mM phenylmethylsulfonylfluoride, chymostatin (10 [micro]g/mL), leupeptin (10 [micro]g/mL), antipain (10 [micro]g/mL), and pepstatin A (10 [micro]g/mL)).