EXOSOME ISOLATION (PLASMA, URINE)
SubX®-Exo-DNA kit is designed for simultaneous isolation of exosomes and circulating cell-free DNA (cfDNA) directly from liquid biopsies (plasma or serum) without Proteinase K. Our proprietary bi-functional substance (SubX®) binds DNA under physiological conditions (e.g. directly in biological liquids) with high affinity degree, followed by adsorption of the [DNA- SubX®] complex on a solid phase matrix. SubX® captures DNA via its phosphate groups and allows for elimination of bias related to both AT/GC content and DNA fragments length, thus improving extraction efficacy and accuracy of downstream applications. Since our method does not require chaotropic salts for DNA capture, no dilution of starting material (and cfDNA) will take place. Both ends of SubX® molecule display phospholipid binding groups. This feature allows each molecule of SubX® to anchor two exosomes (i.e. dimerize and further oligomerize. SubX® oligomerizes exosomes in both highly proteinaceous bioliquids (serum, plasma) and in high salt biofluids (urine, tears, etc.). Excess of SubX® molecules in the solution results in oligomerization of up to 10-15 exosomes and formation of micron-size particles that are easily precipitated in a brief 14K x g centrifugation step. A specially designed buffer allows for reconstitution of the pelleted exosomes back to free monomer format suitable for downstream applications. Different degrees of affinity of SubX® to DNA (strong) and SubX® to exosome phospholipid complexes (mild) allow to separate exosomes and cfDNA from the total SubX® pellet. Exosomes are easily recovered in monomer format by special reconstitute buffer while tightly bound [SubX®-cfDNA-beads] complex remains in pellet and cfDNA further isolated by supplied solutions.
Figure 1. Exosome isolation steps. All procedures after mixing plasma or urine with SubX® are done in one tube. Each incubation takes place at room temperature with occasional vortexing. Exosomes are separated from SubX®-Exosomes pellet by reconstitution in a special buffer ERB.
Figure 2. Simultaneous binding of urine endogenous exosomes and sonicated spike DNA with SubX® followed by sequential isolation of exosomes and then DNA. Profiles of spike DNA (blue) and exosomes (green) before binding with SubX® and after dissociation (pink and red) perfectly coincide.
Figure 3. AFM images of exosome solution adhered on mica after 1 min incubation at room temperature. Exosomes, isolated by ultracentrifugation (A), ultracentrifugation in 30% sucrose (B), agglutination with Concanavalin A (C), isolation with HansaBioMed ImmunoBeads (D), and precipitation with SubX® (E). Scale bar is 300 nm. The pseudo color ruler indicates the particles’ height (nm). The characteristic particle profile of exosomes isolated with SubX® is shown in panel (F), where “h” is exosome height (nm) and “l” – diameter (nm).
Figure 4. Electron microscopy image confirms membrane structure and size distribution of exosomes. Scale bar is 100 nm.
Figure 5. Snapshot images of SubX-bound and reconstituted exosomes taken from NanoSight NS300 video. Exosomes bound to SubX form large complexes with size range from >300 nm to 1200 nm, while after reconstitution in special buffer ERB the size is in the range 50-120nm.
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