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.