bsa pbs t  (Thermo Fisher)

Journal: ACS Chemical Neuroscience

Article Title: Differentiating Alzheimer’s Aβ Isoforms Coaggregated in Cerebrospinal Fluid via Single-Particle Imaging

doi: 10.1021/acschemneuro.5c00692

Figure Lengend Snippet: AFM and super-resolution (STORM) imaging of separately and coaggregated Aβ-40 and Aβ-42 peptides incubated in PBS. (A) AFM topographic image showing the presence of spherical particles of varying sizes and short fibrils adsorbed on a gold substrate. (B) Cross-sectional height profile indicated by the blue arrow in panel A. (C) Super-resolution fluorescence microscopy image of Aβ-40 labeled with Alexa Fluor 561 showing the presence of mainly spherical oligomeric particles. Data shown in panels A–C are based on Aβ-40 peptides incubated separately. (D) AFM image of Aβ-42 protein aggregates showing mostly elongated fibrils together with spherical particles. (E) Cross-sectional height profile indicated by the blue arrow in panel D. (F) Super-resolution fluorescence microscopy image of Aβ-42 labeled with Alexa Fluor 647, detecting both fibrillar and oligomeric protein aggregates. Data shown in panels D–F are based on Aβ-42 peptides incubated separately. (G) AFM image of coaggregated Aβ-40 and Aβ-42 showing the presence of both fibrillar and spherical particles. (H) The cross-sectional height profile is indicated by the blue arrow in panel G. (I) Super-resolution fluorescence microscopy image of Aβ-40 labeled with Alexa Fluor 561 in green and Aβ-42 labeled with Alexa Fluor 647 coded in red. (J) Percentage area and standard deviation taken up by signal from each channel in 4 images of coaggregated samples: Aβ-40 (mean = 0.22 ± 0.14%). and Aβ-42 (mean = 0.47 ± 0.14%). (K) Quantification of spherical particle and fibril height for Aβ-40 and Aβ-42 aggregated independently and coaggregated based on AFM images. (L) ThT kinetics assay of the total concentration of 1 μM of peptides in PBS per sample for independent and coaggregated protein samples.

Article Snippet: Samples were fixed at room temperature for 20 min in 4% paraformaldehyde in PBS, washed 3× with PBS, and blocked in BlockAid ( B10710 , Thermo Fisher) for 1 h. Both primary and secondary antibodies were diluted in 1% BSA in PBS (37525, Thermo Fisher) at a dilution of 1:500.

Techniques: Imaging, Incubation, Fluorescence, Microscopy, Labeling, Standard Deviation, Concentration Assay

Journal: ACS Chemical Neuroscience

Article Title: Differentiating Alzheimer’s Aβ Isoforms Coaggregated in Cerebrospinal Fluid via Single-Particle Imaging

doi: 10.1021/acschemneuro.5c00692

Figure Lengend Snippet: Ion-specific modulation of Aβ 40 –Aβ 42 coaggregate morphology and energetics in PBS versus CSF. (A) Distribution of maximum vertical heights of the coaggregated complex relative to the gold surface in PBS (black) and CSF (red), extracted from the final 100 ns of simulations. A marked leftward shift in CSF indicates reduced oligomer protrusion and enhanced surface adhesion. (B) Average asphericity (Δ) of Aβ 40 oligomer, showing increased sphericity in CSF, consistent with morphological compaction. (C) Domain-resolved interaction energy decomposition between the Aβ 40 oligomer and the Aβ 42 fibril for both PBS and CSF conditions, highlighting altered binding patterns across fibril regions (N-term, CHC, C-term). (D) Representative snapshot of the Aβ 40 –Aβ 42 complex in PBS at the gold–solution interface. Aβ 40 is shown in red, Aβ 42 in blue, ions as spheres, and the Au(111) surface in yellow. (E) Representative snapshot in CSF, showing tighter packing of Aβ 40 . Inset reveals persistent bridging of a Ca 2+ ion between Glu11 (Aβ-40) and Glu22 (Aβ-42), stabilizing the interface. (F) Oligomer–ion and fibril–ion interaction energies (normalized per ion) in PBS and CSF, showing significantly stronger binding of Ca 2+ to both components in CSF. (G) Two-dimensional free energy surface (FES) plots for Aβ-40 asphericity versus total interaction energy in PBS (left) and CSF (right), illustrating distinct morphodynamic regimes: broad, deep-binding states in PBS vs compact, kinetically trapped states in CSF.

Article Snippet: Samples were fixed at room temperature for 20 min in 4% paraformaldehyde in PBS, washed 3× with PBS, and blocked in BlockAid ( B10710 , Thermo Fisher) for 1 h. Both primary and secondary antibodies were diluted in 1% BSA in PBS (37525, Thermo Fisher) at a dilution of 1:500.

Techniques: Binding Assay