Abstract
The ability to isolate and observe molecular changes in protein composition and function at synapses is important in understanding the disease mechanisms. Because signal transmission is highly regulated by transient phosphorylation of neuronal proteins at the synapse, preservation of this protein modification during synaptosome preparation is essential. Therefore, enriched preparations of synaptic particles called synaptosome are necessary to study synapse function. Because of insufficiency of ample sample for quantitative and qualitative analysis via old method, we applied some modifications that were resultant in high synapse yield. Interestingly, we found that modified methods produced more protein as well as more clear protein band on electrophoresis. Therefore, the modified procedure was better than the older method in effort to isolate more pure synapse protein for improved result outcome.To advance the method for our study, the following modifications were made to the regularly used protocols:•The pellet consisting of synaptosomes was cleaned two to three times in HEPES buffer containing proteases inhibitor and centrifuged at 12,000×g for 15min each. This step is highly essential to remove any contamination of sucrose-HEPES buffer and other organelle's which interfere with protein purification analysis.•Following this step, the synaptosome pellets were suspended in RIPA buffer (mixed with protease inhibitor and PMSF) along with 0.2% TritonX-100 and further centrifuged at 20,000×g.•Further, the resulting pellet was discarded and suspended in RIPA buffer (mixed with protease inhibitor and PMSF) only. The sample was immediately used for protein estimation and protein electrophoresis.
Highlights
The method depicts the modified method of synapse protein preparation
To advance the method for our study, the following modifications were made to the regularly used protocols: The pellet consisting of synaptosomes was cleaned two to three times in HEPES buffer containing proteases inhibitor and centrifuged at 12,000 Â g for 15 min each
The synaptosome pellets were suspended in Radio immune assay precipitation buffer (RIPA) buffer along with 0.2% TritonX-100 and further centrifuged at 20,000 Â g
Summary
1. Sterile surgical instrument to harvest brain. Sodium phosphate buffer for washing of brain sample. Normal saline was prepared by dissolving 0.9% of sodium chloride (NaCl), i.e. 9 g of NaCl in 100 ml of distilled water. To prepare the 0.1 M sodium phosphate buffer, 0.2 M of NaOH was added to 250 ml of 0.02 M NaH2PO4Á2H2O to adjust pH to 8.0 and volume made up to 500 ml with distilled water (DW). To prepare the sodium phosphate buffer of 0.03 M, pH 7.0. 150 ml of 0.1 M sodium phosphate buffer (pH 8.0) was taken and pH was adjusted to 7.0 and volume made up to 500 ml with DW.
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