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
(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
Elastase purified from human neutrophils and chymostatin
were from Calbiochem (San Diego, CA).
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)).