Extracellular Vesicle Analysis Through Automated Western Blotting
Extracellular vesicles (EVs) and exosomes have been shown to facilitate intercellular communication and are involved in numerous important cellular & disease processes, making them an exciting potential source for biomarker discovery and for liquid biopsy analysis. While EV protein and exosome characterization is important, analyzing protein expression in intact EVs can be a challenge due to the limited amount of material and low expression levels of many EVs proteins.
Fast, Validated EV Protein Analysis Workflow
QIAGEN and ProteinSimple™, a Bio-Techne brand — leaders in exosome isolation and high sensitivity western blotting solutions respectively — have now teamed up to create a new, validated workflow to address these challenges.
Now you can easily perform western blotting-based protein analysis measurements in isolated intact EV samples using a validated workflow utilizing the exoEasy Maxi Kit by QIAGEN and Simple Western™ automated western blotting instrument by ProteinSimple. Stop struggling with complex workflows using ultracentrifugation or precipitation-based methods for EV isolation or with the qualitative and low sensitivity nature of traditional western blotting workflows!
For EV researchers in academic or industry labs who want to analyze protein expression in EVs via western blotting, this tested solution represents an easy, fast, and high sensitivity workflow that combines high purity EV isolation with reproducible, sensitive western blotting data.
This combined workflow unites the advantages of both technologies resulting in:
- Easy, simple workflow for extracellular vesicle analysis which utilizes a simple column combined with plug and play, hands-free, automated western blotting analysis
- Standardized exosome isolation and protein analysis
- Low sample volume requirements which are ideal for your QC workflows
- High sensitivity detection of low abundance EV proteins with conventional antibodies
- Acceleration of exosome biomarker discovery studies, exosome characterization research and liquid biopsy analysis
High Performance Workflow for Extracellular Vesicle Analysis
EV Protein Expression Across Plasma Fractions Using the exoEasy Maxi Kit and Simple Western
EV eluate of plasma (3 µL) compared to unprocessed plasma and the unbound fraction found in the column flow-through. EV marker Flotillin-1 (Ab133497; Abcam) is not detectable in neat plasma but is detectable in EV eluate, while HDL-marker ApoA1 (AF3664-SP; R&D Systems) is depleted in EV eluate compared to plasma and flow-through fraction.
Detection of MISEV Recommended EV Protein Markers using Automated Western Blotting
The “Minimal Information for Studies of Extracellular Vesicles” (MISEV) guidelines recommend characterizing preparations for trans-membrane-, cytosolic- and contaminating non-EV proteins. However, compliance with these guidelines can be a considerable effort due to lack of easy and robust analytical protocols and the time consuming and user variable nature of standard western blotting protocols. In this virtual poster presentation, we present a simple method for isolation of EVs and analyzing MISEV-recommended protein expression using Simple Western for automated protein separation and immunodetection.
Learn About Validated Assays Based on MISEV Guidelines for EV Proteins and Non-EV Protein Controls
Watch Dr. Chris Heger from ProteinSimple and Dr. Johan Skog from Exosome Diagnostics discuss some of the latest diagnostic applications in the exosome field, and how this validated workflow can help streamline your exosome characterization research.
Simple Western automated western blotting systems from ProteinSimple are hands-free automated capillary electrophoresis (CE)-based platforms which offer high sensitivity and eliminate many of the tedious, error-prone steps of traditional western blotting, making them particularly well-suited for extracellular vesicle analysis. The systems automate protein separation and immunodetection and offer the following advantages for extracellular vesicle analysis:
- Low sample volume: Only 3 µL starting volume required, enabling you to utilize <5% of your EV sample volume for protein expression characterization and the rest for other isolation or characterization methods (e.g., functional testing, nucleic acid isolation, electron microscopy, nanoparticle tracking, etc.)
- Industry-leading sensitivity: chemiluminescence or fluorescence detection with Stellar™ NIR/IR Modules
- Multiplexed protein analysis and RePlex™ sequential immunoassays or total protein detection
- Fully quantitative data with built-in total protein normalization in the same capillary
- Hands-free automation of 24 samples in one 3-hour run
The exoEasy Maxi Kit from Qiagen, Powered by the Exosome Experts at Exosome Diagnostics
The exoEasy Maxi Kit from QIAGEN utilizes membrane affinity spin columns and specialized buffers to efficiently isolate and purify EVs (including exosomes) from plasma, serum, cell culture supernatant and other biological samples. The technology was developed with Exosome Diagnostics, Inc and offers several advantages for intact EV isolation including:
- Consistent isolation of intact exosomes and other EVs
- Suitable for downstream functional testing including physical and biochemical analysis of EVs
- Fast, 25-minute workflow
- Process multiple samples in parallel using a simple spin-column
- Accommodates large sample volumes including up to 4 mL of plasma or serum or 32 mL of cell culture supernatant
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- Protocol: Examining Exosomes on Simple Western PDF
- Auto-Immune Disease, Advantages of Exosome Diagnostics with Saliva Testing
- Genomic Profiling, Advantages of Liquid Biopsy-Based Biomarkers
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