Metabolic labeling experiments at isolated synapses
Metabolic labeling experiments at isolated synapses
mRNA and rRNA are present within dendrites and axons (VanMinnen 1994; Steward 1997). It is puzzling whether mRNAs located in regions outside the cytosol are actually translated. The following approach demonstrates that neural protrusions can indeed synthesize proteins independently of the cytosol.
Modern Neuroscience Research Techniques
Author: U. Windhorst & H. Johansson Translated by Zhao Zhiqi Chen Jun
Operation method
Metabolic labeling experiments at isolated synapses
Materials and Instruments
Fixative Warming solution Chloramphenicol Radiographic self-developing emulsion Developer SDS sample buffer Move I. Radiation autoradiography Neurons were cultured in conditioned medium for 2d as described in Chapter X. The neurons were then incubated in the conditioned medium for 2d. 1. Separate the neural protrusions from the cytosol with a sharp microelectrode and move the cytosol out of the petri dish with a traction electrode (see Chapter X). 2. Block mitochondrial protein synthesis by adding a final concentration of 0.l mmol/L chloramphenicol to the culture medium. 3. Carefully remove the culture medium and wash with warming solution 6 times, each time with Imin. ensure that the axon is always covered with a small amount of liquid; once dry, the axon will disintegrate. 4. Label for 30 min in the incubation solution containing 0.5 mCi/ml trans-35S and 0.1 mmol/L chloramphenicol. 5. Lacquer was washed 6 times for 40 min each with warming medium containing lmol/L unlabeled cysteine and methionine and 0.1_d/L chloramphenicol. 6. Fix the lacquer with 1% polymethylene glycol and 1% acetic acid for 2 hours. 7. Dehydrate with a series of increasing concentrations of ethanol. 8. Dry in air. 9. Immerse in radiographic self-developing emulsion. 10. Expose for 2 to 7 hills depending on the rate of 35S labeling of the incorporated protein. 11. Develop in D19. 12. Short rinse in water. 13. 20% Na2S2O3 (sodium thiosulfate) aqueous solution fixed. 14. Wash the paint with water for 20 min. 15.Seal the film with sealing liquid or glycerin. 1. Separate the neural protrusions from the cytosol and decapsulate the cytosol. 2. 0.1 mmol/L gentamicin was used to pre-warm the isolated neural protrusions for 30 min. 3. Wash the lacquer with the incubation solution 6 times, each time Imin. 4. Labeling for 2.5 h in the incubation solution containing 0.5 mCi/ml trans-35S and 0.1 mmol/L chloramphenicol. 5. Wash with warm solution 6 times for 30 min each time. 6. 20ul of SDS sample buffer was collected from the neural protrusions and transferred into a microcentrifuge tube. 7. Boil for 5 min. 8. Centrifuge the sample at 12OOOr/min for 2 min. 9. The supernatant was spiked to 8% SDS-PAGE for electrophoresis. 10. 50% methanol and 10% acetic acid fix the gel for 30 min. 11. Process the gel with an amplifier for 20 min. 12. Thousand dry the gel. 13. Expose to X-ray film (KodakBiomaxMS) for 1~3 weeks. 14. Developed film To understand the sites of protein synthesis within neural protrusions and the heterogeneity of proteins synthesized at these sites, we applied the trans-35St mnemonic technique to label isolated neural protrusions by 35S-labeled cysteine and methionine. If protein synthesis does occur in isolated axons, then these radiolabeled amino acids will be able to incorporate into the protein and be detected by radioautography. The results demonstrate that neural protrusions are capable of synthesizing a wide variety of proteins and that this process occurs predominantly in varicosities and growth cones (Fig. 3-6). For more product details, please visit Aladdin Scientific website.
