, 2',7'-dichlorofluorescin diacetate, lipopolysaccharides (LPS), 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl-tetrazolium bromide (MTT), Nonidet P-40, phenylmethylsulfonyl fluoride (PMSF), RA, sodium fluoride, sodium chloride, sodium phosphate, Tris-HCl, Triton X-100, and Tween-20 were purchased from Sigma-Aldrich (USA).
25% sodium deoxycholate, and 1% NP40) containing protease inhibitors (1 mmol/L phenylmethylsulfonyl fluoride, 1 mg/ml aprotinin, 1 mg/ml leupeptin
, and 1 mg/ml pepstatin; Roche).
0) containing leupeptin
(10 [micro]g/ml), aprotinin (10 [micro]g/ml), Triton X-100 (1%), PMSF (1 mM), EGTA (1 mM), NaF (5 mM), and sodium orthovanadate (10 mM).
5 [micro]g/mL of leupeptin
, 1 mM of phenylmethylsulfonyl fluoride, and 10 [micro]g/mL of aprotinin), and 1 mM of sodium orthovanadate.
1% sodium dodecyl sulfate (SDS), and 1 mM PMSF, aprotinin (1 [micro]g/mL) and leupeptin
5% sodium deoxycholate, 10 mg/ml aprotinin, 10 mg/ml leupeptin
, and 1 mM phenylmethylsulfonylfluoride.
1 mM EDTA, 1 [micro]g/[micro]l leupeptin
, 1 [micro]g/[micro]l pepstatin, 1 [micro]g/[micro]l aprotinin).
This natural inhibitor is called leupeptin
and has several interesting properties that may be exploited in the field of natural immunotherapy with particular reference to viral infections.
2-mercaptoethanol, acetic acid, hydrochloric acid, phosphoric acid, casein, gelatin, tyrosine, 2-propanol, leupeptin
, phenylmethylsulfonyl fluoride (PMSF), sodium dodecyl sulfate (SDS) and bovine serum albumin (BSA) were purchased from Sigma-Aldrich (St.
Samples was homogenized in protein extraction buffer [2 mM EDTA, 100 uM phenylmethylsulfonyl fluoride (PMSF),10 ugmL-1 pepstatin A , 330 mM sorbitol, 10 ug/mL leupeptin
and 50 mM HEPES/KOH pH 8.
75 [micro]M aprotinin, 1[micro]M leupeptin
, 1[micro]M pepstatin, and 0.
25% sodium deoxycholate, 150 mM NaCl, 1 mM ethylene glycol bis(2-aminoethyl ether)tetraacetic acid (EGTA), 1 mM sodium orthovanadate, 10 mM sodium fluoride, 1 mM phenylmethyl-sulfonyl fluoride, 10 [mu]g/mL aprotinin, 5 [mu]g/mL leupeptin
, and 1 [micro]g/mL pepstatin) for 30 min at 4[degrees]C.