Method and validation of synaptosomal preparation for isolation of synaptic membrane proteins from rat brain

Pradip Kumar Kamat, Anuradha Kalani, Neetu Tyagi

Research output: Contribution to journalArticlepeer-review

30 Scopus citations

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 15 min 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.

Original languageEnglish (US)
Pages (from-to)102-107
Number of pages6
JournalMethodsX
Volume1
Issue number1
DOIs
StatePublished - 2014

Keywords

  • Brain
  • Neurons
  • Synapse protein

ASJC Scopus subject areas

  • Clinical Biochemistry
  • Medical Laboratory Technology

Fingerprint Dive into the research topics of 'Method and validation of synaptosomal preparation for isolation of synaptic membrane proteins from rat brain'. Together they form a unique fingerprint.

Cite this