NAME: I.D.

#: COURSE CODE:

LAB PARTNER: DATE:

TITLE OF LAB: ASSAY OF TISSUE GLYCOGEN MATERIALS: Heart, liver, muscle and kidney from freshly killed rats/guinea pigs (fed and fasted). KOH (300g/L) Saturated Na2SO4 ETOH - 95% (v/v) 2.4M HCl 1.0M NaOH Stock glucose solution (0.2 mol/mL) Dissecting instruments Reagents for the estimation of glucose: Glucose oxidase 12.5 mg Peroxidase o-dianisidine HCl - 1% (w/v) in EtOH All of the above was dissolved in: 0.5M sodium phosphate buffer, pH 7.2 100mL 4.0 mg 0.5 mL

PROCEDURE: The complete heart, X grams of muscle, Y grams of liver and Z grams of kidney were removed from both fed and fasted rats or guinea pigs. After weighing the tissue, it was placed in a tube containing KOH (300g/L).The mixtures were then heated in a boiling water bath for 20 minutes with shaking taking place occasional. The tubes or beakers were the left on ice after this period expired. 2ml of fed liver, fasted liver and two of the three remaining tissues(either the fasted or fed)were collected separately in small test tubes.0.2mL saturated Na2SO4 was added to the tubes which were then vortex well.5mL ethanol (EtOH) (95% v/v) was added to the tubes to precipitate the glycogen. The tubes were allowed to stand for 5 minutes after which they were centrifuged at 3000rpm for 10 minutes. The pellets of precipitated glycogen were retained while the supernatant was discarded. The pellets were dissolved in 5mL distilled water while gently warming the tubes in a water bath set at 37C. The dissolved glycogen from the liver of the fasted animal was poured into a 10mL measuring cylinder. The tube was then rinsed with 2mL distilled water which was transferred to the measuring cylinder. Distilled water was added to the measuring cylinder to bring the volume up to 10mL which was then mixed thoroughly. The dissolved glycogen from the liver of the fed animal was poured into a 100 mL measuring cylinder. The tube was then rinsed with 2mL distilled water which was transferred to the measuring cylinder. Distilled water was added to the measuring cylinder to bring the volume up to 100mL which was then mixed thoroughly. The dissolved glycogen from the heart, muscle or kidney from fed and fasted animals was poured into a 25 mL measuring cylinder. The tube was then rinsed with 8mL distilled water which was transferred to the measuring cylinder. Distilled water was added to the measuring cylinder to bring the volume up to 25 mL which was then mixed thoroughly. Two 1mL aliquots of each of the prepared glycogen was pipette into separate test tubes. 1mL of 2.4M HCl was added to each test tube which was then mixed. All the tubes were well covered with foil and the placed in a boiling water bath for 1 hour. After this time had expired, the solution was neutralized by carefully adding a specific volume of 1M NaOH .the neutralized samples were made up to 5 mL with distilled water. 0.3mL and 0.5mL of the liver or muscle (for either fed or fasted animal) samples were pipette into separate test tubes.0.5 and 1.0mL of the heart and kidney samples were pipette into separate test tubes. The volume was made up to 1.0mL with distilled water where necessary.

2.5mL of enzyme cocktail was added to each tube which was then incubated at 37 C for 45 minutes. The tubes were allowed to cool to room temperature and then r the absorbencies of each at 450nm were read and recorded. The stock glucose solution (0.2 moles/mL) was pipette in the following volumes; 0.0, 0.2 ,0.2, 0.4, 0.6 ,0.6 and 1.0mL , into 7 separate test tubes. The volume in each tube was made up to 1.0ml using distilled water. 2.5mL of enzyme cocktail was added to each tube which was then incubated at 37 C for 45 minutes. The tubes were allowed to cool to room temperature and then the absorbencies of each at 450nm were read and recorded. REFFERENCES: