Experiments on the identification of the Golgi apparatus

Summary

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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