aβ40 peptides  (Sangon Biotech)

Journal: RSC Advances

Article Title: Graphene field-effect transistor based multiplexed sensing platform for simultaneous detection of multiple Alzheimer's disease biomarkers

doi: 10.1039/d5ra07384g

Figure Lengend Snippet: Mixed sample detection results. (a) Higher concentration of Aβ42 biomarker vs. lower concentration of hsa-miR-125b biomarker. The absolute concentrations were (Aβ42 : 125b): 10 pM : 1 fM (10 000 : 1); 50 nM : 50 pM (1000 : 1); and 100 nM : 1 nM (100 : 1). (b) Higher concentration of hsa-miR-125b biomarker vs. lower concentration of Aβ42 biomarker. The absolute concentrations were (125b : Aβ42): 100 nM : 1 fM (10 8 : 1), 100 nM : 1 pM (10 6 : 1), and 50 nM : 50 pM (10 3 : 1). (c) Fixed concentration of Aβ40 biomarker and varying concentrations of hsa-miR-125b ( n ≥ 13, n is number of sensors used for each measurement).

Article Snippet: Aβ42 and Aβ40 peptides, hsa-miR-125b RNA sequence, the hsa-miR-125b complementary DNA probe and the Aβ42 DNA aptamer were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

Techniques: Concentration Assay, Biomarker Discovery

Journal: bioRxiv

Article Title: Medin drives Aβ40 to adopt Aβ42-like fibril polymorphs in vitro

doi: 10.1101/2025.07.17.665283

Figure Lengend Snippet: (A) Thioflavin T (ThT) fluorescence kinetics of Aβ40 (20 µM), medin (20 µM), and an equimolar mixture in 50 mM Tris, 100 mM NaCl, pH 7.4 at 30 °C. Aβ40 aggregates with a lag phase of ∼23 h, while medin aggregates more rapidly (∼3 h). The Aβ40–medin mixture shows an intermediate lag time (∼6.5 h), indicating that medin accelerates Aβ40 nucleation. (B) LC-MS chromatograms from monomeric and post-aggregation samples. Distinct peaks for Aβ40 (6.5 min) and medin (6.9 min) disappear after 5 days, consistent with full incorporation into fibrils. Upon HFIP solubilisation, both peptides are recovered, confirming their presence in the fibrils. (C) Negative-stain TEM images. Aβ40 forms heterogeneous ribbon-like fibrils; medin forms morphologically diverse filaments lacking clear crossover. In contrast, Aβ40–medin fibrils display well-defined helical twist and reduced morphological heterogeneity. (D) Dual immunogold labeling of fibrils using anti-Aβ (6E10; 6 nm gold red asterisk) and anti-medin (1H4; 10 nm gold, green asterisk). Aβ40 and medin fibrils alone show exclusive labeling. In the mixture, fibrils are labeled with 6E10, 1H4, or both, indicating formation of homotypic (Supplementary figure 2) and heterotypic assemblies. (E) Representative cryo-EM micrograph of Aβ40–medin fibrils and their 2D class averages showing two distinct populations: Population A (wide, twisted fibrils with ∼100 nm crossover) and Population B (narrow, straight fibrils with ∼60 nm crossover). A pie chart displays the relative distribution of both morphologies based on the number of extracted particles used for helical reconstruction. Aβ40-alone samples did not yield fibrils suitable for helical reconstruction.

Article Snippet: Aβ40 peptide was obtained from rPeptide (Catalog ID: A-1153) in HFIP-treated film form.

Techniques: Fluorescence, Liquid Chromatography with Mass Spectroscopy, Staining, Labeling, Cryo-EM Sample Prep

Journal: bioRxiv

Article Title: Medin drives Aβ40 to adopt Aβ42-like fibril polymorphs in vitro

doi: 10.1101/2025.07.17.665283

Figure Lengend Snippet: (A) Cryo-EM density map of Population A fibrils (right) and a transverse section showing a four-layered cross-β arrangement (left). (B) Transverse section of the 3D cryo-EM map of Population A fibrils, fitted with two inner β-sheets composed of extended β-strands spanning residues Q15 to V40. (C) Surface representation of the atomic model of Population A, highlighting the location of residues in the aggregation-prone region and the interface between the two protofibrils. (D) Structural comparison of the atomic model of Population A with previously reported Aβ40 fibril structures. Top: Cross-sectional alignment of Population A with fibril morphologies from PDB IDs 8ff3, 8ot1, and 6w0o. Bottom: Side view alignment. In the Population A model, side chains are represented in stick format, whereas reference fibrils display only the peptide backbone, each in a unique color as indicated.

Article Snippet: Aβ40 peptide was obtained from rPeptide (Catalog ID: A-1153) in HFIP-treated film form.

Techniques: Cryo-EM Sample Prep, Comparison

Journal: bioRxiv

Article Title: Medin drives Aβ40 to adopt Aβ42-like fibril polymorphs in vitro

doi: 10.1101/2025.07.17.665283

Figure Lengend Snippet: (A) Cryo-EM density map of Population B fibrils (left) and a transverse section of the cryo-EM map highlighting two F-shaped subunits (top right). Cartoon representation of a single F-shaped subunit with the three β strands labelled as β1, β2 and β3 (Bottom right). (B) Transverse section of the 3D cryo-EM map of Population B fibrils, fitted with two F-shaped subunits composed of Aβ40 (D1–V40). These subunits face each other, forming the protofibril interface through interactions at their C-termini. Unresolved densities on both sides of the fibrils are highlighted with a red box. (C) Surface representation of the atomic model of the Population B fibril, highlighting the staggered polar arrangement of the two protofibrils. Key residues, including the amyloid-prone region KLVFFA, are annotated with sequence labels. (D) Key stabilizing interactions that contribute to the unique architecture of Population B fibrils. (Top left) cryo-EM map of C-terminal residues 28–40 (GAIIGLMVGGVV), forming a hydrophobic loop with V40, I32, and M35 buried in the core. (Bottom left) cryo-EM map of the N-terminal polar cluster formed by residues H6, S8, and Y10. (Right) Interaction of D1 from one protofibril with E22 and D23 of a nearby subunit from the opposite protofibril, forming a charge cluster. A possible metal ion location is highlighted by a red asterisk.

Article Snippet: Aβ40 peptide was obtained from rPeptide (Catalog ID: A-1153) in HFIP-treated film form.

Techniques: Cryo-EM Sample Prep, Sequencing

Journal: bioRxiv

Article Title: Medin drives Aβ40 to adopt Aβ42-like fibril polymorphs in vitro

doi: 10.1101/2025.07.17.665283

Figure Lengend Snippet: (A) t-SNE projection of Aβ fibril polymorphs based on per-residue FoldX energy profiles. Each point represents a published Aβ40 or Aβ42 fibril structure or a structure from this study (Pop A and Pop B). Colors indicate isoform and origin (e.g., in vitro, ex vivo, seeded). Population A clusters tightly with canonical in vitro Aβ40 structures. In contrast, Population B groups with a subset of Aβ42 fibrils, including 8olg (from APP23 mouse brain) and 5oqv (seeded Aβ42 fibrils), indicating conformational similarity. These fibrils share features such as a structured N-terminus, buried hydrophobic C-terminal loop, and polar, non-planar subunit geometry. Despite being composed of Aβ40 sequence, the Population B morphology mimics key structural characteristics of Aβ42 fibrils and supports the view that heterotypic interaction with medin can shift Aβ40 folding toward an Aβ42-like energetic and architectural state. (B) Structural alignment of the F-subunit (violet) with the L-S subunit (grey) of in vitro produced Aβ42 (PDB ID: 5oqv). The two subunits are perfectly aligned from the C-terminus up to His14; deviation begins at His13 (highlighted by a red box).

Article Snippet: Aβ40 peptide was obtained from rPeptide (Catalog ID: A-1153) in HFIP-treated film form.

Techniques: Residue, In Vitro, Ex Vivo, Sequencing, Produced

Journal: bioRxiv

Article Title: Midkine Attenuates Aβ Fibril Assembly and Amyloid Plaque Formation

doi: 10.1101/2025.03.20.644383

Figure Lengend Snippet: a , Protein sequences of MDK proteins expressed in mammalian 293 or E. coli cells. The N-terminal His-tag was cleaved after expression by TEV protease. b, Workflow of bottom-up LC-MS/MS analysis to characterize the positions of disulfide (S-S) bonds. Proteins were subjected to IAA alkylation, trypsin digestion and LC-MS/MS analysis. Crosslinked peptides containing single or double S-S bonds were identified. c, Relative peak intensities of top five crosslinked peptides ( n = 3 replicates). d-e, Effect of 293- or E. coli -expressed MDK (10 µM) on Aβ40/42 (5 µM) fibrillation kinetics in ThT fluorescence assays. Results represent the average of 3 replicates.

Article Snippet: Subsequently, 10 μM of the 15 N-labeled Aβ42 and Aβ40 peptides were incubated with 0, 5, and 10 μM MDK in low-binding tubes (Eppendorf, #0030108434) at 37°C for 24 and 48 h, respectively.

Techniques: Expressing, Liquid Chromatography with Mass Spectroscopy, Fluorescence

Journal: bioRxiv

Article Title: Midkine Attenuates Aβ Fibril Assembly and Amyloid Plaque Formation

doi: 10.1101/2025.03.20.644383

Figure Lengend Snippet: a , Highly purified recombinant MDK proteins expressed in mammalian 293 or E. coli cells, visualized on a stained SDS gel, with molecular weight (MW) markers. b , Schematic representation of top-down LC-MS for analyzing disulfide bond formation in MDK proteins. Free thiol (-SH) groups in Cys residues are alkylated by iodoacetamide (IAA), introducing a mass shift that differentiates them from proteins with intact disulfide bonds (S-S). The AlphaFold-predicted MDK structure (containing 5 S-S bonds) is downloaded, highlighting the N-terminus (N), C- terminus (C) and only one S-S bond (in red). c , Intact mass raw spectrum and deconvoluted results of 293-expressed MDK, revealing different charge states for a single protein form with 5 S-S bonds. d , Intact mass raw spectrum and deconvoluted results of E. coli -expressed MDK, showing charge states for three protein forms containing 5, 4, or 3 S-S bonds. The relative abundance of these forms was quantified from three independent scans, with data presented as mean ± SEM. e-f , Effect of 293- or E. coli -expressed MDK (3 µM) on Aβ40/42 (5 µM) fibrillation kinetics in ThT fluorescence assays. Results represent the average of 3 replicates.

Article Snippet: Subsequently, 10 μM of the 15 N-labeled Aβ42 and Aβ40 peptides were incubated with 0, 5, and 10 μM MDK in low-binding tubes (Eppendorf, #0030108434) at 37°C for 24 and 48 h, respectively.

Techniques: Purification, Recombinant, Staining, SDS-Gel, Molecular Weight, Liquid Chromatography with Mass Spectroscopy, Fluorescence

Journal: bioRxiv

Article Title: Midkine Attenuates Aβ Fibril Assembly and Amyloid Plaque Formation

doi: 10.1101/2025.03.20.644383

Figure Lengend Snippet: a, ThT fluorescence assay detecting Aβ40 amyloid fibril formation with titrated MDK protein ( n = 3 replicates, averaged data shown). b , Analysis of secondary nucleation rate constant ( k 2 ) by globally fitting the ThT data using AmyloFit software , estimating k 2 at each MDK concentration in a unit (M -2 h -1 ), followed by log transformation and linear fitting of these values at different MDK concentrations. c , CD spectroscopy of Aβ40 (10 µM) with or without MDK, reporting ellipticity in millidegrees (mdeg). d , ThT assay of Aβ42 and MDK ( n = 3 replicates, averaged data shown). e , k 2 analysis for Aβ42 fibrillation using AmyloFit. Data points from the 10 µM MDK condition did not fit well and were excluded from the fitting. f , CD spectroscopy of Aβ42 (10 µM) with or without MDK. g-h , Negative stain EM of Aβ40/MDK and Aβ42/MDK samples, respectively, with the scale bars shown.

Article Snippet: Subsequently, 10 μM of the 15 N-labeled Aβ42 and Aβ40 peptides were incubated with 0, 5, and 10 μM MDK in low-binding tubes (Eppendorf, #0030108434) at 37°C for 24 and 48 h, respectively.

Techniques: Fluorescence, Software, Concentration Assay, Transformation Assay, Circular Dichroism, ThT Assay, Staining

Journal: bioRxiv

Article Title: Midkine Attenuates Aβ Fibril Assembly and Amyloid Plaque Formation

doi: 10.1101/2025.03.20.644383

Figure Lengend Snippet: a, Diagram of the secondary nucleation model illustrating Aβ assembly. The rate constants ( k + and k 2 ) are defined with units, reflecting their dependency on protein concentration and time. b-c, Analysis of Aβ40 and Aβ42 elongation constants ( k + ) by fitting the ThT data. d , Effect of MDK (0-3 µM) on Aβ40 fibrillation kinetics ( n = 3 replicates, averaged data shown). e, Effect of MDK (0-3 µM) on Aβ42 fibrillation kinetics ( n = 3 replicates, averaged data shown). f, Time course CD spectra of Aβ40 alone (0-24 h). g, Time course CD spectra of Aβ40 with MDK (0-24 h). h, Time course CD spectra of Aβ42 alone (0-24 h). i, Time course CD spectra of Aβ42 with MDK (0-24 h).

Article Snippet: Subsequently, 10 μM of the 15 N-labeled Aβ42 and Aβ40 peptides were incubated with 0, 5, and 10 μM MDK in low-binding tubes (Eppendorf, #0030108434) at 37°C for 24 and 48 h, respectively.

Techniques: Protein Concentration, Circular Dichroism

Journal: bioRxiv

Article Title: Midkine Attenuates Aβ Fibril Assembly and Amyloid Plaque Formation

doi: 10.1101/2025.03.20.644383

Figure Lengend Snippet: a , Purified Ub protein on a stained SDS gel. b-c, Top-down mass spectrum and deconvoluted results of Ub protein showing different charge states. d, Bottom-up LC-MS/MS analysis of Ub, covering the full Ub sequence except two short tryptic peptides (in grey). e, ThT fluorescence assay measuring Aβ40 fibril formation at different Ub concentrations ( n = 3 replicates, averaged data shown). f , CD spectroscopy of Aβ40 with or without Ub, with ellipticity reported in millidegrees (mdeg). g , Negative stain EM of Aβ40/Ub samples with a scale bar (100 nm). h , ThT fluorescence assay measuring Aβ42 fibril formation at different Ub concentrations ( n = 3 replicates, averaged data shown). i. CD spectroscopy of Aβ42 with or without Ub. j, Negative stain EM of Aβ42/Ub samples with a scale bar (100 nm).

Article Snippet: Subsequently, 10 μM of the 15 N-labeled Aβ42 and Aβ40 peptides were incubated with 0, 5, and 10 μM MDK in low-binding tubes (Eppendorf, #0030108434) at 37°C for 24 and 48 h, respectively.

Techniques: Purification, Staining, SDS-Gel, Liquid Chromatography with Mass Spectroscopy, Sequencing, Fluorescence, Circular Dichroism

Journal: bioRxiv

Article Title: Midkine Attenuates Aβ Fibril Assembly and Amyloid Plaque Formation

doi: 10.1101/2025.03.20.644383

Figure Lengend Snippet: a, 1 H- 15 N HSQC spectra of Aβ40 (10 μM) with MDK at 0, 5, or 10 μM in 50 mM Tris buffer (pH 7.5). Spectra were collected before incubation (left panel) and after 48 h incubation (right three panels). b, Relative cross-peak intensities for each residue, excluding D1 and H14. Intensities were normalized to a maximum value of 1. c, Relative percentage of Aβ40 intensities after 48 h incubation compared to pre-incubation values. Percentages were calculated pre residue and averaged, with data shown as mean ± SEM. Statistical significance was analyzed by two-tailed Student’s t -test. d-f, HSQC spectra of Aβ42 under similar conditions as Aβ40, but with 24 h incubation. Statistical significance was analyzed by two-tailed Student’s t -test.

Article Snippet: Subsequently, 10 μM of the 15 N-labeled Aβ42 and Aβ40 peptides were incubated with 0, 5, and 10 μM MDK in low-binding tubes (Eppendorf, #0030108434) at 37°C for 24 and 48 h, respectively.

Techniques: Incubation, Residue, Two Tailed Test

Journal: bioRxiv

Article Title: Midkine Attenuates Aβ Fibril Assembly and Amyloid Plaque Formation

doi: 10.1101/2025.03.20.644383

Figure Lengend Snippet: a, 1 H- 15 N HSQC spectra of Aβ40 (10 μM) with or without Ub (10 μM) in 50 mM Tris buffer (pH 7.5). Spectra were collected before incubation (left panel) and after 48 h incubation (right two panels). b, Relative cross-peak intensities for each residue, excluding D1 and H14. Intensities were normalized to a maximum value of 1. c-d, NMR analysis of Aβ42 under similar conditions as Aβ40, but with 24 h incubation.

Article Snippet: Subsequently, 10 μM of the 15 N-labeled Aβ42 and Aβ40 peptides were incubated with 0, 5, and 10 μM MDK in low-binding tubes (Eppendorf, #0030108434) at 37°C for 24 and 48 h, respectively.

Techniques: Incubation, Residue

Journal: bioRxiv

Article Title: Midkine Attenuates Aβ Fibril Assembly and Amyloid Plaque Formation

doi: 10.1101/2025.03.20.644383

Figure Lengend Snippet: a , The diagram of the Mdk gene knockout and PCR genotyping of the WT and homogenous Mdk KO mice. The Mdk gene consists of five exons, including the first exon as the 5’ untranslated region. A CRISPR-mediated deletion was introduced within exon 3, disrupting the open reading frame and resulting in a gene knockout. Crossbreeding of Mdk KO with FAD mice generated four genotypes for comparison. b , Western blot analysis confirming the loss of MDK protein and increased Aβ in the brain of heterogenous FAD mice with homogenous Mdk KO (12-month-old, n = 3 replicates). The Aβ intensity was quantified from the western blot image and normalized by setting the mean value of FAD mice to 1. c , Differential extraction to generate Sarkosyl-soluble and -insoluble fractions of mouse brains. d , ELISA analysis of Aβ40 and Aβ42 in the Sarkosyl-soluble and -insoluble fractions of mouse cortices ( n = 10 per group), with the unit of ng protein per mg brain tissue. The Sarkosyl-insoluble fractions were further dissolved in an 8 M urea-containing buffer and diluted for ELISA. e , Quantification of X34-positive amyloid plaque in the cortices (FAD n = 10, FAD/KO n = 11). The relative levels of area were calculated as the percentage of the total plaque area within the entire cortical region and normalized by setting the mean value of FAD mice to 1. f , Quantification of microglia density and area in the cortices (FAD n = 10, FAD/KO n = 11). g , Co-immunofluorescence staining of amyloid plaque, MDK and IBA1 in the mouse cortices. Scale bar, 50 µm. Statistical significance was analyzed using a two-tailed Student’s t -test if the two groups had equal variances, or by Welch’s t -test if the two groups had unequal variances. Data are shown as mean ± SEM. in c-f. Each point represents a data point of one mouse. Full statistical information is in Source Data Statistics.

Article Snippet: Subsequently, 10 μM of the 15 N-labeled Aβ42 and Aβ40 peptides were incubated with 0, 5, and 10 μM MDK in low-binding tubes (Eppendorf, #0030108434) at 37°C for 24 and 48 h, respectively.

Techniques: Gene Knockout, CRISPR, Generated, Comparison, Western Blot, Extraction, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining, Two Tailed Test

Journal: bioRxiv

Article Title: Midkine Attenuates Aβ Fibril Assembly and Amyloid Plaque Formation

doi: 10.1101/2025.03.20.644383

Figure Lengend Snippet: a , Predicted mouse MDK protein sequences in WT and CRISPR-mediated KO mice, with N-terminal signal peptide shown in green. The 23 base pair deletion in MDK gene alters the open reading frame in the KO mice, potentially generating a truncated protein with distinct C-terminus (in blue) from WT. Peptides detected by MS in brain proteomic analysis are also shown, with shared peptides between MDK and the potential truncated form highlighted in red. The two peptides (aa 30-47 and aa 31-47) allow quantification of MDK full length proteins and the tentative N-terminal truncation. b , Quantification of two shared peptides in FAD and FAD/KO. The intensities were normalized by setting the highest value to 100. The absence of their signals in FAD/KO mice strongly suggests that the tentative N-terminal truncation was not expressed at a detectable level. c , Expression of Mdk , Gfap (astrocyte marker) and Pdgfra (oligodendrocyte progenitor cell marker) in mouse brain, shown in Uniform Manifold Approximation and Projection (UMAP) plots. The cell-type clusters were derived from single-cell RNA sequencing of more than 1.2 million cells in the whole cortex and hippocampus using the 10x Genomics Chromium platform . Expression levels are indicated by color intensity (red). Mdk is highly expressed in the 377_Astro cluster, with a trimmed mean (25-75%) Log₂(CPM+1) value of 4.13, as well as in the 366_Oligo cluster, with a trimmed mean value of 0.72. d , ELISA analysis of Aβ40/Aβ42 ratio in the Sarkosyl-soluble and -insoluble fractions of mouse cortices ( n = 10 per group). e , Quantification of X34-positive amyloid plaque density in the cortices of male (FAD n = 5; FAD/KO n = 5) and female (FAD n = 5, FAD/KO n = 6). f , Quantification of X34-positive amyloid plaque area in the cortices of the same sets of male and female mice. The results were normalized by setting the mean value of FAD mice to 1. Statistical significance between FAD and FAD/KO in male or female was analyzed by two-tailed Student’s t -test. g , Example images of X34 staining showing amyloid plaque quantification in the cortices of FAD and FAD/KO mice. Cortices were outlined by the white lines. Scale bar, 500 µm. Data are shown as mean ± SEM in panels b , d , e , f . Full statistical information is available in Source Data.

Article Snippet: Subsequently, 10 μM of the 15 N-labeled Aβ42 and Aβ40 peptides were incubated with 0, 5, and 10 μM MDK in low-binding tubes (Eppendorf, #0030108434) at 37°C for 24 and 48 h, respectively.

Techniques: CRISPR, Expressing, Marker, Derivative Assay, RNA Sequencing, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Staining