8. Transfer the gel from casting plate onto a UV-transparent thick plastic sheet and place it in a staining tray containing ethidium bromide solution for 20-30 min. 9. For destaining the gel, place it in water for 15-20 min. 10.
The solution was cooled to a lower temperature and mixed with 2µl of EtBr. This solution was poured into a trough which contained a comb, for polymerization. Then this stacked gel was placed in the tank which contained TBE buffer and then the comb was removed carefully for the formation
In the round-bottom flask (100 mL), we placed p-aminobenzoic acid (1.2 g) and ethanol (12 mL). We swirled the mixture until the solid dissolved completely. We used Pasteur pipet to add concentrated sulfuric acid (1.0 mL) to the flask. We added boiling stone and assembled the reflux. Then, we did reflux for 75 minutes.
Wash with 95% ethanol and wash with water after 15 seconds. 7. Stained counterstain with fuchsine and wash with water after 60 seconds. 8. Remove excess water from the surface of the gram stained glass slide and observed under 1000X oil immersion microscope.
3.10.2Column Packing Before starting to pack a column, a small piece of cotton is gently will insert into the centre hole of column with the aid of a long stick. A small amount of n-hexane will add into the column. Silica gel weighted is mixed with n-hexane to form a mobile slurry solution and then slowly transferred into column. The side of the column will gently tape to prevent the formation of air bubbles in the column. Additional solvent will use to add the remaining silica gel into the column.
Formula 2: % Component= 100% component mass (g) sample mass (g) Procedure First, we measured out the evaporating dish to find the mass. Then we added around 3 grams of our sample (2.832g exactly). Next we added the isopropyl alcohol to dissolve the Benzoic Acid. We filled the evaporating dish, stirred, and then decanted the sample into a 140mL beaker with a stirring rod. This
An equivalent volume of saline solution was added (NaCl 0.16 M; pH = 7) and it was mixed completely. The resultant solution was centrifugated for 30 minutes at 9000 rpm at 4ºC in a rotor Beckman JA-20, eliminating the pellet. The supernatant was collected to obtain the lipoproteins by gradient density ultracentrifugation: 10ml of the yolk solution were taken and then 0.9 g of potassium bromide (KBr) was added. The bromide was dissolved with a smooth agitation, with care not to denature the proteins. Straight afterwards, saline solution was added (NaCl 0.16 M, pH=7) to the 10ml of the yolk solution with a Pasteur pipette, avoiding the sample diffusion, forming two phases and filling the tube completely.
Embedding was done with two changes of molten paraffin for 30 min to remove xylene. They were mounted in L blocks with molten paraffin and solidified. The solidified blocks were trimmed to small size and sectioned using microtome (Spencer U.S.A) 3 to 5-micron thickness. 5.2.3 Toluidine blue Staining: Toluidine blue is a metachromatic stain that stains nucleus which appears blue in color. The isolated rat's brain was fixed and processed as per the tissue processing procedure, and then brain tissue slices were prepared for toluidine blue staining.
Briefly, the cartridges were preconditioned by flushing with 2 mL of methanol and 1 mL of HPLC water. Separately, 50 µL of plasma sample plus 100 µL of an 85% phosphoric acid:water mixture (1:10) and 10 µL of internal standard solution (diclofenac at 100 µg/mL) were vortex mixeding. Then, samples were loaded into the cartridge and allowed to stand for 5 min, washed with 0.6 mL of a water:methanol mixture (95:5. v/v) and then dried under vacuum. The (S)-ketoprofen was eluted with 1 mL of an acetonitrile:methanol mixture (50:50, v/v) at a flow rate of 1 mL/min. The eluate was evaporated to dryness in a water bath at 37.0 ± 0.5 ᵒC under a gentle stream of nitrogen.
To prepare the column for chromatographic separation, a 5.75 inch Pasteur pipette is required; plugged with glass wool (or cotton) at the bottom. The column was filled up to ½ of the height of the pipette, and then loaded with a thin layer of sand. The sample was loaded on top of the sand and loaded again with a thin layer of sand. Eluents were loaded one after the other: hexane; dichloromethane with hexane; dichloromethane; and dichloromethane with methanol. Elution was the main process used in this experiment; there are two types of elution isocratic and gradient.