Appreciate if you could help me answering the following questions! thank you!!
How would you prepare a 20% homogenate from a 2.0g of liver using 0.25M sucrose?
why is it necessary to cool the homogenate during homogenization?
Sure, I'd be happy to help answer your questions!
To prepare a 20% homogenate using 2.0g of liver and 0.25M sucrose, you would follow these steps:
1. Begin by weighing out 2.0g of liver and placing it in a homogenization tube or vessel.
2. Add an appropriate volume of 0.25M sucrose solution to the liver. To calculate the volume needed, you can use the formula: Volume of sucrose = (Weight of liver × Desired concentration) / Concentration of sucrose. In this case, the desired concentration is 20% or 0.20 and the concentration of sucrose is 0.25M. Plugging those values into the formula, we get: Volume of sucrose = (2.0g × 0.20) / 0.25M = 1.6mL of 0.25M sucrose.
3. Pipette or pour the calculated volume, in this case, 1.6mL, of 0.25M sucrose solution into the homogenization tube containing the liver.
4. Homogenize the liver and sucrose mixture. This can be achieved using a homogenizer or by manually grinding the liver using a pestle or other suitable method. The goal is to break down the liver tissue into smaller, uniform particles.
5. Once the homogenization is complete, the resulting mixture is called a homogenate.
Now, as for your second question, "Why is it necessary to cool the homogenate during homogenization?"
Cooling the homogenate during homogenization is necessary to prevent heat-induced denaturation or degradation of proteins and other heat-sensitive molecules present in the liver tissue. The cooling process helps maintain the integrity and functionality of these biomolecules.
During homogenization, mechanical forces generate heat due to friction. This heat can have detrimental effects on the proteins and enzymes present in the liver tissue. Cooling the homogenate minimizes the increase in temperature and helps to preserve the native conformation and activity of the proteins.
The cooling can be achieved by placing the homogenate on ice or using a temperature-controlled homogenization device. By keeping the temperature low, you can ensure that the homogenate will remain at a suitable temperature to maintain the integrity of the cellular components.