This protocol allows for the extraction of the liver glycogen molecules in a manner that preserves their structure and limits the amount of the small glycogen particles lost. With previous research showing diabetes alter glycogen structure, this method could provide in studying to diabetes and the various glycogen storage disease. To extract glycogen from the liver, start by transferring one gram frozen liver tissue to a 15 milliliter tube containing six milliliters of glycogen isolation buffer.
While keeping on ice, homogenize the liver tissue using a tissue homogenizer. When done, transfer half or three milliliters of the cell suspension to a new tube, followed by boiling for 10 minutes. Keep the other half of the suspension on ice to extract glycogen-containing associated proteins that are not denatured.
For the glycogen content determination, remove an eight microliter aliquot from each tube and keep the tube on ice. After spinning the remaining suspension at 6, 000 G for 10 minutes at four degrees Celsius, transfer the supernatant to ultracentrifuge tubes to centrifuge them at 3.6 times 10 to the fifth G and four degrees Celsius for 90 minutes. After discarding the remaining supernatants, resuspend the pellets in 1.5 milliliters of glycogen isolation buffer.
Then, layer the samples over 1.5 milliliters of 30%sucrose solution in four milliliter ultracentrifuge tubes, followed by centrifugation as described before for two hours. After discarding the supernatants, resuspend the pellets in 200 microliters of deionized water. To precipitate glycogen from the cell suspension, add 800 microliters of absolute ethanol to the cell suspensions and mix well.
Then, store the mixtures at minus 20 degrees Celsius for at least one hour to allow precipitation. Once precipitation occurs, centrifuge the samples at 6, 000 G for 10 minutes at four degrees Celsius and resuspend the glycogen pellet in 200 microliters of deionized water to repeat the ethanol precipitation process thrice. From the final cell suspension, remove an aliquot of eight microliters from each tube for glycogen content determination.
Then, freeze the remaining supernatants in liquid nitrogen and freeze dry overnight. The next day, store the dried glycogen samples in the freezer at minus 20 degrees Celsius. The study analyzed the purity, crude yield, and glycogen yield of the dried glycogen sample extracted by different conditions.
There were no significant differences in crude yield and glycogen yield between the groups extracted with the various conditions. In contrast, the glycogen purity was significantly influenced by the sucrose concentrations and the addition of a boiling step. Glycogen with the highest purity was extracted using the 30%sucrose concentration and a 10-minute boiling step.
Glycogen was extracted from the liver homogenate by either 30%50%or 72.5%sucrose, boiled or unboiled, to assess the effects of the various conditions on the size of the molecules in the final extract. Chain length analysis was performed on six livers for both boiled and unboiled, using 30%50%and 72.5%sucrose concentrations. The content of the glycogen molecules or beta particles with a hydrodynamic radius, RH, lower than 30 nanometers was calculated.
The boiled samples had lower average RH values and a higher particle content than the unboiled samples. Also, lower sucrose concentrations resulted in lower RH values and higher beta particle contents. The introduction of a boiling step also led to lower RH max or the maxima values, while the sucrose concentration had no significant effect.
Make sure tissue is fully homogenized and there are no chunks of the tissue remaining. So, this techniques paves the way for us to explore what paths links the smaller beta particles together to form the larger alpha particles.
