Exosome Proteomics Sample Preparation
1. Sample Collection
First, samples containing exosomes must be collected. This typically involves gathering from sources such as cell culture media, blood, urine, and cerebrospinal fluid, depending on the research objectives. For instance, tumor cell culture media is often collected for cancer studies.
2. Centrifugation and Ultracentrifugation
Once the samples are collected, exosomes are isolated through a series of centrifugation and ultracentrifugation steps. Initially, low-speed centrifugation (300-500g) is used to eliminate cells and cell debris. This is followed by high-speed centrifugation (10,000-20,000g) to remove smaller particles. Finally, ultracentrifugation (100,000-200,000g) is employed to collect the exosomes.
3. Washing and Re-centrifugation
To remove potential interfering proteins and lipids, the exosome pellet is washed with sterile PBS, followed by another round of ultracentrifugation.
4. Dissolution and Protein Extraction
The exosome pellet is dissolved using an appropriate buffer, such as RIPA or SDS, and proteins are extracted using standard methods. These proteins are then suitable for downstream proteomic analyses, including LC-MS/MS analysis or Western blotting.
5. Protein Quantification
Quantification of the samples is necessary to ensure uniform protein concentration across all samples. This can be achieved using BCA, Bradford, or other established protein quantification methodologies.
Outlined below are the essential steps for preparing exosome proteomics samples. Adherence to these steps ensures the acquisition of high-quality exosome protein samples, which provide a robust foundation for subsequent proteomics analyses.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
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