Briefly, 1 ml of suspension medium was taken from the 10% tissue homogenate. 0.5 ml of 30% Trichloroacetic acid (TCA) was added to it, followed by 0.5 ml of 0.8% thiobarbituric acid (TBA) reagent. The tubes were covered with aluminium foil and kept in shaking water bath for 30 minutes at 80°C. After 30 minutes, tubes were taken out and kept in ice-cold water for 30 minutes. These were centrifuged at 3000 rpm for 15 minutes.
1ml of the homogenisation buffer was added to the P1 pellet and was vortexed to resuspend it. The supernatant was then removed from the P2 tube and placed into a micro tube labelled ‘S’. 1 ml of the homogenisation buffer was added to the P2 pellet and placed on ice. The pellet was then resuspended again by adding small quantity of glass beads and it was vortexed vigorously until the pellet has disappeared from the bottom of the
The sample was transferred to a 250 ml conical flask kept in water bath for alkali treatment. 75 ml of 17.5% caustic soda was measured using a measuring cylinder at 20°C. 15 ml of 17.5% NaOH was added and fibres were macerated gently with a flattened glass rod for 1 minute. 10 ml more NaOH was added and the solution was mixed for 45 seconds. 10 ml NaOH was again added and mixed for 15 seconds to make lump free slurry.
H2SO4 • Catalyst: potassium sulphate (K2SO4), copper (II) sulphate pentahydrate (CuSO4.5H2O) • NaOH (40%) • 0.1 N HCl solution • Boric acid (4%) • Indicator: methyl red (200 mg make up to 100 ml with %95 ethanol ) 1 gram Feta Cheese was used as a sample in this experiment. First of all, the experiment was started with the digestion phase which was performed by mixing 10g potassium sulphate and 0.5 g CuSO4 solution that acts as catalyst, and added 20ml conc. H2SO4. The digestion unit temperature was set to 420±10°C and preheated for 15 minutes.
22.5 g of plate count agar powder was dissolved in a litre of sterile distilled water on the hot plate 2. pH of the solution was adjusted to 7.0 ± 0.2 by adding NaOH or HCl and was immediately transferred into the Schott bottle to be autoclaved at 121 ° C for 15 minutes 3. Prepared medium was stored in 4° C chiller Lauryl Sulphate Broth
2.5.4. Metal chelating activity Briefly, 2 mM FeCl2 was added to different concentrations of test sample and reaction was initiated by the addition of 5 mM ferrozine. The mixture was vigorously shaken and left to stand at room temperature for 10 min. Absorbance was measured at 562 nm after 10 min.8 % Inhibition = [(AB - AA)/AB] x 100, where AB, absorption of blank sample, AA, absorption of test sample.
Alginate sample (30 mg) was hydrolyzed in 10 mL HCl (0.3 M) at 100 ºC for 2 h. After cooling, the mixture was centrifuged (6000 rpm, 45 min), and the supernatant solution was separated and neutralized with 1 M NaOH and referred to as fraction A. The insoluble material was dissolved in 1 M NaOH and the pH was decreased to 2.85 by the addition of 1 M HCl. The suspension was recentrifuged and the supernatant was separated and referred to as fraction B. The insoluble fraction was dissolved by neutralization with 1 M NaOH and referred to as fraction C. The fractions A, B, and C are enriched in MG, MM, and GG blocks
The DEAE CL-6b gel was washed twice with 0.5m Hcl, twice with 0.5m Naoh and twice with PB ph6.0 before utilization. For DEAE 1ml gel every 1ml serum was utilized. In the wake of blending for 1 hour at 200c the suspension was centrifuged at 4500g for 25 minutes. The supernatant was thought by Ammonium sulfate precipitation and pellet was broken up in refined water and the ensuing arrangement was dialyzed against PBS ph7.2 to evacuate Ammonium sulfate and conformed to the first volume with yhe same cushion (Hong et.al., 1994). 2)
Fe3O4 nanoparticles were synthesized according to our previously reported method by chemical co-precipitation of Fe2+ and Fe3+ ions with a molar ratio of 1:2 . Briefly, 2.4 g of FeCl3.6H2O and 0.8 g of FeCl2.4H2O were dissolved in 30 mL of deionized water under using continuous N2 purge at 70 °C. and Under vigorous stirring, followed by dropwise addition of 10 mL of NH3.H2O was dropwise added to the reaction mixture until the color of mixture turned to black and kept reacting for 90 min to complete the reaction. At the end, the synthesized Fe3O4 nanoparticles were separated by a magnet and washed by using water and ethanol for further three times with 89.3%
Cells were disrupted using RLT buffer and the lysate was homogenized by centrifugation through a QIAshradder spin column for 2 min at full speed. The flowthrough was mixed with 70% ethanol (equal volume as the RLT buffer used), transferred into an RNeasy spin column and centrifuged at 17000 g for 1 min. after having the flowthrough discard, RW1 buffer was added to the column, centrifuged at 17000 g for 1 min and the flowthrough was discard. The spin column was washed twice with RPE buffer under at 17000 g centrifugation for 2 min. The column was centrifuged empty at full speed for 1 min to remove any residual ethanol.
The sample was then incubated at 56°C to lyse the tissue. The sample was checked every fifteen minutes and vortexed between each checking for an hour and a half until the tissue was completely lysed. The tissue sample was then again vortexed. Next 200 microliters of buffer AL was added and
Once the gel hardened, .5X TBE (44.5 mM Tris base, 44.5 mM boric acid, and 1.0 mM EDTA) was added just until the gel was covered with the TBE buffer. Each sample was loaded into the gel as well as 10 μL of DNA size markers (1kb ladder, New England Biolabs) into a separate lane. The gel was allowed to harden at room temperature and then electrophoresed at 100 volts for 75 minutes. Using a UV imager, a photo was taken of the resulting traveled DNA fragments in the gel.