Small Extracellular Vesicles/Exosomes Protein Analysis

Exosomes are rich in proteins, which provide vital information about their origin, function, and characterization. Common methods for detecting exosomal proteins include Western Blotting, Enzyme-Linked Immunosorbent Assay (ELISA), Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS), and Nano Flow Cytometry (NanoFCM). According to the guidelines published by the International Society for Extracellular Vesicles in “MISEV 2018,” it is recommended that exosome protein analysis should ideally include a combination of transmembrane proteins, cytosolic proteins, and negative proteins. The actual detection approach can be optimally chosen based on factors like sample quantity, species-specific antibodies, and instrumentation limitations.

 

Western Blot (WB)

Western blot operates on the principle of antigen-antibody specific binding to detect exosomal marker proteins, identifying exosomes at the protein level. Our company has thoroughly reviewed commonly used antibodies in the literature, and we routinely offer clients a choice of five positive markers(CD9, CD63, CD81, TSG101, Syntenin) and one negative marker(Calnexin) antibody. Considering the immunotyping variability of exosomes, it is possible that the expression level of certain positive markers might fall below the sensitivity threshold of Western blot detection, resulting in no positive bands. To address this, we routinely incorporate positive controls to verify the accuracy of experimental procedures and antibody binding. Moreover, we recommend that our clients consider employing methods with higher sensitivity, such as the Flow NanoAnalyzer and the fully automated protein quantification system (WES), to enhance detection rates.

 

Nano Flow Cytometry (NanoFCM)

Exosomes typically display intact antigens on their surface, allowing for incubation with fluorescence-labeled specific monoclonal antibodies. Subsequently, these exosomes can be detected using NanoFCM. By analyzing the proportion of positive particles detected, the expression of surface proteins on the exosomes can be precisely determined, facilitating the detection of exosomal surface proteins. Common specific marker proteins found in exosomes include CD9, CD63, and CD81. After incubating the exosomes with fluorescently labeled antibodies, excess free antibodies are removed through ultracentrifugation, enabling more accurate detection with NanoFCM(We typically provide three antibody markers for detecting human and mouse proteins; for other species or different surface markers, customers need to provide directly labeled antibodies).

 

Fully Automated Protein Quantification System (WES)

The WES system employs the innovative Simple Western technology, which revolutionizes the traditional Western Blot method that has been a standard for over 30 years. This technology allows for the completion of sample separation, capture, immobilization, immunodetection, and quantitative analysis all within a single capillary. Its advantages include the elimination of gel preparation and electrophoresis, as well as membrane transfer, thus streamlining experimental steps and minimizing sample loss. The system automates both primary and secondary antibody incubation as well as chemiluminescence detection, leading to significantly improved accuracy and repeatability of results. With a sample loading volume of just 3-5 µl, it conserves precious samples and antibodies. The entire process takes only three hours, offering a rapid and efficient detection with high sensitivity, reaching the picogram level. Upon completion of the experiment, the system automatically generates comprehensive analytical data, including protein size, signal-to-noise ratio, peak area, and concentration, facilitating precise quantitative analysis of the results.

 

Services at NextGen Exsome

1.Western Blot Protein Markers of Exosomal Expression Analysis

Detecte the expression of signature proteins in exosomes.

 

Deliverables:

 
1)PVDF film development pictures and white light pictures in TIF format

2)Strip results in PPTX format

 

2.NanoFCM Exosomes Membrane Protein Expression Positivity Rate Analysis

Detect the positivity rate of exosomal membrane protein expression.

 

Deliverables:

 
1)Sample report in PDF format

2)Instrument raw data in FCS format

 

3.WES Protein Markers of Exosomal Expression Analysis

Detecte the expression of marker proteins in exosomes.

 

Deliverables:

 
1)Developed pictures in PDF format

2)Instrument raw data in CBZ format