Experiments on the identification of the Golgi apparatus
Experiments on the identification of the Golgi apparatus
This experiment is based on the "Guide to Cellular Experiments", translated by Huang Peitang et al.
Operation method
Subdivision of the Golgi apparatus by preparative free-flow electrophoresis
Materials and Instruments
Golgi Move 1. Separate the Golgi-rich fraction from the 1.2 mol/L sucrose/homogenate interface to a final volume of about 5 ml. For more product details, please visit Aladdin Scientific website.
Chamber Buffer
2. 3 mg of Aspergillus crassus X-A α-amylase and 3-A α-amylase from barley malt were added separately.
3. Hold the mixture at 4°C for 45 minutes.
4. At the end of the holding time, the suspension was blown 40 times with a Pasteur pipette with an inner diameter of about 1 mm at the tip to complete the process of destacking.
5. Prepare the electrophoresis medium (Chamber buffer).
Chamber buffer:
10 mmol/L Diethanolamine
10 mmol/L iconic acid
5 mmol/L glucose
0.25 mmol/L sucrose
0.5 mmoI/L MgCl2
6. The following conditions are used in electrophoresis (unless otherwise recommended by the manufacturer): 167 mA (constant current), 131 ± 10% V/cm, buffer flow rate of 2.75 ml per division per hour, sample injection rate of 3.5 ml/hour, temperature of 6°C, and a continuous free-flow electrophoresis unit (e.g., VAP 21, Weber GmbH, Munich, Germany). .
7. The resulting fractions were collected by centrifugation, e.g. in a microcentrifuge for 2 min, and then resuspended and used for analysis.