sheep polyclonal nrxn3  (R&D Systems)

Journal: bioRxiv

Article Title: Identification of markers for the isolation of neuron-specific extracellular vesicles

doi: 10.1101/2024.04.03.587267

Figure Lengend Snippet: Validation of NRXN3 as neuron-specific marker present on EVs and immuno-isolation of NRXN3 + EVs. A. Simoa measurement in SEC fractions of pooled CSF of NRXN3 (top), CD9 (middle) and albumin (bottom). B. Simoa measurement in SEC fractions of pooled plasma of NRXN3 (top), CD9 (middle) and albumin (bottom). C. Simoa measurement in DGC fractions of pooled plasma of NRXN3 (top), CD9 (middle) and albumin (bottom). D. Simoa measurement of NRXN3 in SEC fractions of plasma from three different individuals. For A-D, Simoa measurements were taken in duplicate for each sample. Error bars indicate SD.

Article Snippet: The following antibodies were used for immuno-isolation of NRXN3 and controls: sheep polyclonal NRXN3 (cat. AF5269, R&D Systems), GFP (4745-1051, Rockland), and Isotype Control (AB37385, Abcam).

Techniques: Biomarker Discovery, Marker, Isolation, Clinical Proteomics

Journal: bioRxiv

Article Title: Identification of markers for the isolation of neuron-specific extracellular vesicles

doi: 10.1101/2024.04.03.587267

Figure Lengend Snippet: Development and validation of EV immuno-isolation method and application to NRXN3. A. Schematic of immuno-isolation of EVs enriched by ultracentrifugation from the cell culture media of K562 cells. B. Western blotting of CD81 after immuno-isolation of EVs. Equal amounts of EVs obtained by differential ultracentrifugation of K562 cell culture media were loaded as input or immuno-isolated using either an anti-CD81 antibody or a control non-specific antibody. PD = pulldown, FT = flow-through C. Schematic of EV immuno-isolation experiment from human plasma or CSF for western blotting analysis. EVs are immuno-isolated from biofluid without initial EV purification. Immuno-isolation was performed using a target antibody or control antibody (PD1 = pulldown 1) and the flow-through is subjected to a second immuno-isolation with the target antibody (PD2 = pulldown. 2) D. Western blots of CD9 (top), CD63 (middle), or CD81 (bottom) after immuno-isolation from human CSF. In each case, the first pulldown (PD1) was performed with antibodies against the target protein or a control antibody, and the second pulldown (PD2) was performed with antibodies against the target protein using the flow-through of PD1. E. Western blots of CD9 (top), CD63 (middle), or CD81 (bottom) after immuno-isolation from human plasma. In each case, the first pulldown (PD1) was performed with antibodies against the target protein or a control antibody, and the second pulldown (PD2) was performed with antibodies against the target protein using the flow-through of PD1. F. Schematic of EV immuno-isolation experiment from human plasma or EVs enriched by SEC from human plasma for assessment by Simoa in the flow-through (FT). G. Simoa measurements of CD9 (left), CD63 (middle), or CD81 (right) of the flow-through of human plasma after immuno-isolation with antibodies against CD9, CD63, CD81 or GFP (non-specific control). H. Simoa measurements of CD9 (left), CD63 (middle), or CD81 (right) of the flow-through of EVs enriched from human plasma by SEC after immuno-isolation with antibodies against CD9, CD63, CD81 or GFP (non-specific control). For G and H, two separate replicates of all samples were used and Simoa measurements were taken in duplicate for each sample. Error bars indicate SD. I. Schematic of NRXN3 immuno-isolation experiment. NRXN3 in pulldown fraction (PD) was measured using Western blotting. NRXN3 in flow-through (FT) was measured using Simoa. Immuno-isolation experiment was performed using antibody against target (NRXN3) or control (GFP or isotype control) J. Western blotting of NRXN3 after pulldown from CSF using either NRXN3 antibody or control antibody (GFP or isotype control). Simoa measurement of NRXN3 in flow-through after pulldown from CSF using either NRXN3 antibody or control antibody (GFP or isotype control). K. Western blotting of NRXN3 after pulldown from plasma using either NRXN3 antibody or control antibody (GFP or isotype control). Simoa measurement of NRXN3 in flow-through after pulldown from plasma using either NRXN3 antibody or control antibody (GFP or isotype control). For J and K, Simoa measurements were taken in duplicate for each sample. Error bars indicate SD.

Article Snippet: The following antibodies were used for immuno-isolation of NRXN3 and controls: sheep polyclonal NRXN3 (cat. AF5269, R&D Systems), GFP (4745-1051, Rockland), and Isotype Control (AB37385, Abcam).

Techniques: Biomarker Discovery, Isolation, Cell Culture, Western Blot, Control, Clinical Proteomics, Purification