Fig 1.
Molecular structures of di-succinimidylsuberate (disuccinimidyl suberate, DSS) and bis-succinimidylsuberate sodium salt (Bis(sulfosuccinimidyl, BS3).
Fig 2.
Aβ monomers cross-linked with glutaraldehyde and BS3.
(A) Aμ1–42 and (B) Aμ1–40 monomer cross-linked with glutaraldehyde under ice bath conditions. The final concentrations of glutaraldehyde were 0.3 mM, 0.6 mM and 1.2 mM, and 1 minute, 5 minute and 10 minute incubation periods were used. (C) Aμ1–42 and (D) Aμ1–40 monomer cross-linked with BS3 under ice bath conditions. The final concentration of BS3 were 0.3 mM, 0.6 mM and 1.2 mM, and 1 minute, 5 minute and 10 minute incubation periods were used. A 5 μM final concentration of Aβ was used in all the glutaraldehyde and BS3 crosslinking experiments. A total of 1 μg sample was added to each electrophoresis lane, and the 16.5%Tris-Tricine gel was analyzed using silver staining. The concentration of Aβ42 (E) and Aβ40 (F) were the same as the crosslinking concentration. Samples were incubated in an ice bath for 1, 5, or 10 min.
Fig 3.
Aβ1–42 monomers, ADDL and protofibrils were cross-linked with glutaraldehyde or BS3.
All experiments were carried out under ice bath conditions. The samples were analyzed on a 16.5% Tris-tricine gel. (A) In the absence of crosslinking, three forms of Aβ1–42. Aβ1–42 was crosslinked with 0.3 mM glutaraldehyde for 1 minute.Aβ1–42 was cross-linked with 0.3 mM BS3 for 5 minutes, and 1 μg sample was added to each electrophoresis lane. The gels were analyzed using silver staining. The "←" indicates the position of the aggregates in the protofibril lane. (B) The same crosslinking reaction conditions as “(A)” were used. But in "(B)", the amount of protein used in each electrophoresis lane was 10 ng. The results were analyzed using the murine monoclonal antibody, 4G8. (C) Histogram showing the grayscale value of each band in (A). “Aggregates” indicates the sum of all other bands excluding monomers. (D) Histogram showing the grayscale value of each band in (B).
Fig 4.
BS3 crosslinking to analyze the behavior of Aβ1–42 interaction with liposomes
(A) The Aβ1–42 monomer was incubated with liposomes in the absence of crosslinking. The samples were assessed by electrophoresis and silver stain analysis. There was no significant difference when comparing the Aβ1–42 monomer with "Aβ1–42 monomer + liposome". (B) The Aβ1–42 monomer was added to liposomes, and the mixture was crosslinked with 3 mM BS3 for 5 minutes under ice bath conditions. Compared with the Aβ1–42 monomer, the "Aβ1–42 monomer + liposome" precipitate, but not the supernatant samples, clearly formed oligomers. A total of 1 μg of sample was added to each electrophoresis lane. (C) All experimental conditions were the same as "B", but the amount of protein used in each electrophoresis lane was 10 ng. The results were analyzed by a murine monoclonal antibody, 4G8, and Western blot. (D) Aβ1–42 DPPC was incubated with liposomes for 2 hours. The staff was 200 nm, and the liposomes had a diameter of 300 nm. After discarding the Aβ1–42 fiber, the samples were centrifuged and resuspended. The samples without crosslinking were assessed by TEM. (E) All experimental conditions were the same as "(D)", but the sample was crosslinked with 3 mM BS3 before TEM sample preparation. The staff was 100 nm.
Fig 5.
Electron microscope photographs of Aβ1–42 monomer, ADDL and protofibril.
(A), (B) and (C) show the non-crosslinked samples. (D), (E) and (F) show the samples that had been crosslinked with 0.3 mM BS3 for 5 minute. (G), (H) and (I) are samples that had been crosslinked with 0.3 mM glutaraldehyde for 1 minute. The staff was 50 nm.
Fig 6.
Analysis of BS3-Aβ crosslinking by SEC on a Superdex 75 10/300 GL SEC column.
(A) Aβ40 monomer (red line: DMSO solubilization; dark blue line: 6 M guanidine-HCl solubilization; sapphire line: 0.3 mM BS3 crosslinking; black line: 0.3mM glutaraldehyde crosslinking). Fifty microliters was used for each sample (Aβ40 concentration: 150 μM). (B) Aβ40 protofibrils (red line: no crosslinking; sapphire line: 0.3 mM BS3 crosslinking; pink line: 0.3 mM glutaraldehyde crosslinking). Aβ40 monomer (black line: 6 M guanidine-HCl solubilization). Fifty microliters was used for each sample (Aβ40 concentration: 300 μM). (C) Analysis of the peak area for the elution of monomeric Aβ40 (crosslinked or not crosslinked) from monomer samples. Each sample was injected three times. For each sample analysis, the total (aggregate + monomer) peak area was expressed as 100%. For Aβ40 monomers without crosslinking, the monomer:aggregate ratio was 93%:7%. For Aβ40 monomers with BS3 crosslinking, the monomer:aggregate ratio was 84.87%:15.13%. For Aβ40 monomers with glutaraldehyde crosslinking, the monomer:aggregate ratio was 72%:28%. (D) Analysis of the peak area for the elution of protofibril Aβ40 (crosslinked or not crosslinked) from monomer samples. Each sample was injected three times. For each sample analysis, the total (aggregate + monomer) peak area was expressed as 100%. For Aβ40 protofibrils without crosslinking, the monomer:aggregate ratio was 85.3%:14.7%. For Aβ40 protofibrils with BS3 crosslinking, the monomer:aggregate ratio was 75%:25%. For Aβ40 protofibrils with glutaraldehyde crosslinking, the monomer:aggregate ratio was 69.6%:30.4%.