Mwco slide a lyzer mini dialysis unit

Human serum samples from patients with PDAC (stage III or IV) and negative controls were collected by Oregon Health & Science University (OHSU), USA. All procedures were in accordance with the Institutional Review Board of Oregon Health and Science University approval. The serum proteins were biotinylated, according to a previously described protocol^{18 (link),19} . Biotinylation is widely used for protein applications, such as immobilization and functionalization^{16 (link),18 (link),20 (link),21 (link)} and to verify that biotinylation of the serum sample did not considerably result in misrepresentation of the actual protein distribution we performed an analysis, using immunoprecipitation and mass spectrometry. The results showed a similar number of matching peptides for the target antigen both before and after biotinylation. In brief, 5 µL of serum samples were diluted 1:45 in phosphate-buffered saline (PBS) and labeled with 0.6 mM EZ-Link Sulfo-NHS-LC-Biotin (Thermo Fisher Scientific). Unbound biotin was removed by dialysis against PBS for 72 h using a 3.5 kDa MWCO Slide-A-Lyzer MINI dialysis unit (Thermo Fisher Scientific), changing buffer every 24 h. The labeled serum samples were aliquoted and stored at −80 °C. In the assay, 75 µL of streptavidin-coated magnetic beads, Dynabeads M-280 (Life Technologies), were used to immobilize and display 1 µL of biotinylated serum proteins.

For virus labeling, 100 μl of purified virus was incubated with 50 μg of AlexaFluor488-amine reactive dye (Invitrogen) at 4 °C for 2 h. To remove all unbound fluorescence, labeled viruses were dialyzed against PBS (containing 1 mM EDTA) in a MWCO Slide-A-Lyzer MINI dialysis unit (Thermo Scientific) at 4 °C overnight39 (link). Tissues were removed from uninfected ferrets. The Alexa488-labeled NC/02 and NC/02^HA149 viruses were added (100 HAU/ml) and incubated at 4 °C overnight. The tissues were formalin-fixed paraffin embedded, deparaffinized with xylene, and hydrated with alcohol. To visualize the cell nuclei, sections were counterstained with DAPI (Invitrogen) and attached virus viewed under a Zeiss LSM510 laser scanning confocal microscope40 (link).

Peak fractions from the glycerol-gradient analyses of the pre-termination, PEC–Integrator–PP2A–SOSS and free Integrator–PP2A–SOSS complexes were separately cross-linked using 0.2% (v/v) glutaraldehyde for 10 min on ice. The cross-linking reaction was quenched using 100 mM Tris-HCl (pH 8) for 10 min on ice. The cross-linked cryo-EM samples were dialysed for 4–6 h against a buffer containing 20 mM HEPES pH 7.4, 150 mM NaCl, 1% (v/v) glycerol, 3 mM MgCl₂, 1 mM DTT and 0.01% (w/v) CHAPS using a 20 kDA MWCO Slide-A-Lyzer MINI Dialysis Unit (Thermo Fisher Scientific). A 2.6–2.8-µm-thin carbon film was floated on the dialysed cryo-EM samples and incubated for 5–15 min depending on the concentration of the sample before cross-linking. The floated carbon film was transferred onto a Quantifoil R3.5/1 copper mesh 200 grid and instantly blotted for 2 s with blot force 5 before being vitrified in liquid ethane using a Vitrobot Mark IV (Thermo Fisher Scientific). The vitrobot was operated at 4 °C and 95–100% humidity.