The extracted heart was homogenized in an ice-cold buffer containing 250 mM sucrose, 10 mM HEPES/1 mM EDTA (pH 7.4), 1 mM Pefabloc (Roche, Switzerland), 1 mM EDTA, 1 mM NaF, 1 [micro]g/mL aprotinin, 1 [micro]g/mL leupeptin, 1 [micro]g/mL pepstatin
, 0.1 mM bpV(phen), and 2 mg/mL glycerophosphate.
In order to detect cytochrome c, lysing was performed on ice for 30 min in a buffer, consisting of HEPES (20 mM), NaCl (500 mM), MgCl2 (1.5 mM), 20 % glycerol, 0.1 % triton X100, EDTA (0.2 mM), aprotinin (1.7 mg/mL), antipain (1.5 mM), pepstatin
(1 mM), dithiothreitol (DTT, 1 mM), phenylmethylsulfonyl fluoride (PMSF, 0.5 mM), and 0.5 % sodium dodecyl sulfate (SDS).
Treated and control epimastigotes parasites from both strains were centrifuged at 3000 x g for 5min at 4[degrees]C, the pellet was washed three times with tryps wash buffer (100 mM NaCl, 3mM MgC[l.sub.2], 20 mM Tris-Hcl, pH 7.5) and incubated in lysis buffer (7M Urea, 2M Thiourea, 4% CHAPS, 40 mM Tris, 0.1 mg/mL PMSF; 1M DTT, 1 mg/mL Pepstatin
, 10 mg/mL Aprotinin and 10 mg/mL Leupeptin; all enzyme inhibitors from Sigma-Aldrich) for two hours.
Finally, to inhibit aspartic protease activity, 29 mM Pepstatin
A dissolved in dimethyl sulfoxide (DMSO) was used.
Von Der Helm, "Human immunodeficiency virus has an aspartic-type protease that can be inhibited by pepstatin
A," Proceedings of the National Acadamy of Sciences of the United States of America, vol.
A30-1 lipase -- Staphylococcus aureus SDS Lipase L2 EDTA, PMSF, pepstatin
A, BME, DTT TA lipases Chemicals with References tolerance Pseudomonas sp.
Proteins were extracted from cultured Caco-2/TC7 cell monolayers using RIPA buffer (150 mmol/L NaCl, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% sodium dodecyl sulfate, 50 mmol/L Tris HCl pH 8.0, 0.2 mmol/L phenylmethylsulfonyl fluoride, 1 [micro]g/mL pepstatin
, 0.5 [micro]g/mL leupeptin), and 50 [micro]g of total protein was used for immunoblotting.
The inhibitors tested were Piper essential oils (48 [micro]g/mL) and 10 mM Pepstatin
A (standard inhibitor).
They are inhibited by a hexapeptide from Streptomyces that contains two statin residues called pepstatin
. Aspartic proteases are also sensitive to diazoacetyl-DL-norleucine methyl ester (DAN) and 1, 2-epoxy-3-(p-nitrophenoxy) propane (EPNP) in the presence of copper ions.
The ipsilateral cortex tissue of the mouse brain was homogenized in prechilled buffer containing 50 mM Tris-HCl (pH 7.4), 2.0 mM EGTA, 2 mM Na3VO4, 50 mM NaF, 0.5 mM AEBSF, 10 [micro]g/ml aprotinin, 10 [micro]g/ml leupeptin, and 4 [micro]g/ml pepstatin
PICs (e.g., aprotinin, leupeptin, antipain, pepstatin
A, phenylmethylsulfonyl fluoride, EDTA, and thimerosal) can avoid proteolysis through the inhibition of serine-, cysteine-, aspartic-, and metallo-proteases.
The tissue was dissociated using an ultrasonic cell disruptor and lysed in cold lysis buffer containing 10 mM Tris-HCl, pH8.0, 240 mM NaCl, 5 mM EDTA, 1 mM dithiothreitol, 0.1 mM phenylmethanesulfonyl fluoride, 1% Triton X-100, 1 mM sodium vanadate, and 1 g/ml of leupeptin, pepstatin
, and aprotinin.