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
Chamber Buffer

Move

1. Separate the Golgi-rich fraction from the 1.2 mol/L sucrose/homogenate interface to a final volume of about 5 ml.

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.

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