Glycogen extraction ZOO 120

The liver was homogenized using blender. It was then weighed and placed in a 125-mL Erlenmeyer flask.
Afterwards, 30% hot aqueous KOH was added and the resulting mixture was heated in a boiling water
bath for one hour. After heating, the mixture was added with 15 mL distilled water and was transferred
to a 250-mL Erlenmeyer flask. To precipitate the glycogen, 30mL of 95% ethanol was added. The mixture
was allowed to cool to room temperature and stood in an ice bath for 20 minutes. It was then
transferred into centrifuge tubes and centrifuged 5000 rpm for 5 minutes. The precipitate with the
glycogen was collected and dissolved with 6mL of cold 10% TCA solution. The mixture was centrifuged
again for 5 minutes and the supernatant was then collected. To recover the glycogen, 15mL of 95%
ethanol was added. The mixture was placed in an ice bath for 10 minutes and was then centrifuged for 5
minutes to collect the glycogen precipitate. The collected precipitate was dissolved with 10mL absolute
ethanol and added with 8mL distilled water to reprecipitate the glycogen. The mixture was stood in an
ice bath and centrifuged for 5 minutes to collect the precipitate. The precipitate was then washed with
4mL diethyl ether and was allowed to dry in a pre-weighed watch glass. After drying, the isolated
glycogen precipitate was weighed.

For the preparation of the standard curve, 2.0mM of glucose solution was used as the stock solution.
Seven test tubes were then added with different concentration of the stock solution. The amount of
glucose and distilled water (in mL) used were as follows, respectively: T1 – 0.0, 1.0; T2 – 0.1,0. 90; T3-
0.2, 0.80; T4 – 0.4, 0.6; T5 – 0.6, 0.4; T6 – 0.8, 0.2; T7 – 1.0, 0.0.

A 10mg/mL glycogen stock solution was prepared using 50mg of the isolated glycogen added in a 50mL
beaker and diluted with 5mL distilled water. Eight test tubes were used with varying concentration of
glycogen. Test tube 1 had 0.4mL distilled water, test tube 2 had both 0.6mL distilled water and 0. 4mL
glycogen solution, while test tubes 3-8 each had 0.4mL glycogen solution. Afterwards, all test tubes
except test tube 2 were added with 0.60mL of 2N HCl. Test tubes 1 and 2 were finally added with 1mL of
1.2N NaOH solution and were allowed to stand in room temperature. Meanwhile, test tubes 3-8 were
placed in a boiling water bath. Test tubes 3-8 were removed at 5-minute intervals which started from 5
minutes after boiling. Each of test tubes 3-8 was added with 1mL of 1.2N NaOH immediately after
removing from the water bath at a specified time. All the test tubes were then diluted with 8mL distilled
water and were mixed using a vortex mixer. A 0.5 mL aliquot from each of the samples was obtained and
diluted again to a final volume of 1.0 mL using distilled water and was mixed using a vortex mixer.

Both the standard solution and the samples were assayed using Nelson’s assay. For all the test tubes of
the standard and the sample, 1.0 mL of Nelson’s reagent was added. The tubes were covered with
marbles and were placed in a boiling water bath for 20 minutes. They were then cooled to room
temperature and added with 1mL arsenomolybdate reagent. Afterwards, the tubes were mixed using a
vortex mixer and were allowed to stand for 5 minutes at room temperature. And finally, 7mL distilled
water was added to each test tubes and again mixed using a vortex mixer.

Using the spectrophotometer, the absorbance of the standards and the samples were read at 510nm.
The standard curve was graphed by plotting the absorbance at 510 nm versus the glucose concentration.
Meanwhile, the content of the glucose in the hydrolyzed glycogen was obtained through interpolating its
absorbance values on the ẋ of the standard curve.