Ength of 245 mm.
The power output of generator is 80 W. The
Ength of 245 mm. The power output of generator is 80 W. The power output at the acoustic horn was 48.4 W/cm2 with a 50 duty cycle of 0.9 sec on and 0.9 sec off and a frequency of 25.1 kHz. The protein was dissolved in 0.05 M Tris-HCl buffer at pH 7.4, containing 0.15 M NaCl. The 1-Bromo-2-fluoro-4-methoxy-5-nitrobenzene sample solution in a polypropylene tube was placed in a water bath at 37 . The US wire was loaded in sample solution at a depth of 1 cm. During US treatment, temperature increase did not exceed 0.5 .Gel-permeation chromatographyFibrinogenolysis was carried out in 0.05 M Na-phosphate buffer, containing 0.15 M NaCl at pH 7.4. Proteolysis was initiated by addition of 150 l of plasminogen (2 mg/ml) and t-PA (0.2 mg/ml) to 15 ml of the solution of fibrinogen (3 mg/ml). The digestion mixture was then incubated at 37 and samples were collected after PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/12958591 certain time intervals for SDS-PAGE and determination of TCA-soluble peptide analysis.Fibrinolysis Clot preparationChromatography was carried out on an automatic GradiFrac (Pharmacia Biotech) system including a programmed fraction collector, a flow UV-detector (l = 280 nm), a gradient mixer, and a peristaltic pump. A Sepharose 4B-CL column (10 ?950 mm) was used. The buffer containing 0.05 M Tris-HCl at pH 7.4 and 0.15 M sodium chloride was used to equilibrate the column. Different fibrinogen species were eluted at a flow rate 0.1 ml/min.SDS-PAGEThe fibrin clot was prepared by an addition of 300 l of 1 M CaCl2 and 1.5 ml of thrombin solution (25 unit/ml in 0.05 M Tris-HCl, 1-[[(1 0.5 M NaCl at pH 7.4) to 15 ml fibrinogen solution (3 mg/ml in the same buffer). The final mixture was incubated for 2 hour at 37 . The resultant clot was dried between the two sheets of the filter paper.Clot lysisElectrophoresis in 10 and 7.5 polyacrylamide with and without reduction with 5 (v/v) 2-mercaptoethanol was performed as described by Laemmli [31]. ProteinThe clot was placed in the polypropylene tube with buffer, containing 15 ml 0.05 M Tris-HCl and 0.5 M NaCl at pH 7.4. For the initiation of the fibrinolysis 150 l of the plasminogen solution (1.5 mg/ml in 0,025 M TrisHCl, 50 glycerol, pH 7,4) and 150 l rt-PA (10 Ethyl 5-(aminomethyl)furan-2-carboxylate hydrochloride mg/ml in 0.05 M Tris-HCl, 0.5 M NaCl (pH 7.4)) were added to the mixture. The mixture was incubated at 37 and during 4 hours of hydrolysis the samples were collected. The aliquots were used for SDS-PAGE (a mixture for protein denaturation with or without 2-mercaptoethanol was directly added to these aliquots) and the determination of TCA-soluble peptide.Cherniavsky et al. BMC Biochemistry 2011, 12:60 http://www.biomedcentral.com/1471-2091/12/Page 4 ofDetermination of TCA-soluble peptideTo 1.5 ml analyzable sample 1.0 ml 15 TCA were added. Samples centrifuged (8000 ?g, 5 min) and the optical density of solution at 280 nm was measured. The concentration of the TCA-dissoluble peptide they recounted to the appropriate concentration of tyrosine into mole/L according to the formula:Tyr = D280 nm ?2.5/1.Statistical analysisStatistical analysis was carried out employed the Microsoft Excel 2000 software. Results were tested by the Student’s t test. Data expressed as mean +/- standard deviation. A p value 0.05 was considered to be statistically significant.Results Fibrinogen was exposed to ultrasound for 30 minutes at 37 and then assayed by SDS-PAGE under reducing and nonreducing conditions (Figure 1). Gel-electrophoresis without reduction of inter- PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/8833965 and intramolecular disulfide bonds showed presence of one polypeptide withthe molecular w.