Pre-formed Fibrils (PFFs)--A Novel Approach to Modeling Neurodegeneration
Schematic presentation of protein misfolding and aggregation
Protein aggregation is a major pathological feature of neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), and Huntington's disease (HD). Under pathological conditions, Tau, Amyloid beta, alpha-synuclein, TDP-43, Huntingtin and other proteins integrate into a wide array of undesirable structures, ranging from oligomers and prefibrillar assemblies to highly ordered aggregates. The fibrillar structure represents a rapid growth phase of protein aggregation as these fibrils are "active" and rapidly recruit monomers for elongation. In additions, these fibrils randomly break into shorter fragments that could act as "seeds", which transmits to other cells and independently recruits monomers to form novel fibrils. Pre-formed fibrils (PFFs) are in vitro formed active fibrils that have this "seeding" activity and are capable of continuously recruiting soluble endogenous pathological proteins to form aggregates and finally induce neurodegenerative pathologies.
The establishment of reliable disease models is crucial for discovering the pathological mechanisms, evaluating the efficacy of therapeutic interventions, and assessing the safety of drug candidates. Compared with traditional disease modeling approaches, PFFs induced pathology does not depend on gene editing, chemical or physical damage, and it can better mimic naturally occurring pathological states processes. Therefore, PFFs is a novel approach to modeling neurodegenerative diseases.
Pre-formed Fibrils (PFFs) Production and Validation
PFFs could be generated from monomers either by incubating at 37°C and shaking, or by heparin induction. Quality control and precise initial preparation are important for successful experiments. One important aspect of successful induction of PFFs is the use of high-quality monomer with high purity, high concentration and correct conformation. Meanwhile, since the application of PFFs is cellular and animal experiments, the control of endotoxin is also an important factor. In addition, prior to the use of PFFs, PFFs should become a length of 50 nm or shorter via ultrasound to ensure the recruiting activity of PFFs as well as to facilitate the endocytosis of PFFs.
The morphology and activity of PFFs could be verified by electron microscope and thioflavin T (ThT) fluorescence assay respectively. Successfully induced PFFs show fibril structure under electron microscope. ThT assay is a classic assay to detect β-sheet structures. PFFs acquire more and more β-sheet structures as they recruit monomers, and when ThT binds to the β-sheet structure, the fluorescence value increases, thus reflecting the activity of PFFs.
Pre-formed Fibrils (PFFs) Product List
As the brand of ACROBiosystems that focused on neuroscience, Aneuro provides Tau-441 PFFs, alpha-synuclein PFFs, Amyloid beta PFFs, TDP-43 PFFs and SOD-1 PFFs, supporting and accelerating the establishment of reliable neurodegenerative models.
| Molecule | Cat. No. | Product Description | Monomer Source | Expression System | Preorder/Order |
|---|---|---|---|---|---|
| Tau | TAU-H5115 | Human Tau-441/2N4R Pre-formed Fibrils Protein, Tag Free | TAU-H5117 | E.coli | |
| TAU-H5116 | Human Tau-441 K18 Pre-formed Fibrils Protein, Tag Free | TAU-H5114 | E.coli | ||
| TAU-H5113 | Human Tau-441 K18 (P301L) Pre-formed Fibrils Protein, Tag Free | TAU-H5118 | E.coli | ||
| TAU-H511c | Human Tau-381 / 1N3R Pre-formed Fibrils Protein, Tag Free | TAU-H5112 | E.coli | ||
| TAU-H5148 | Human Tau-441 (P301S) Pre-formed Fibrils Protein, His Tag | TAU-H5145 | E.coli | ||
| TAU-H5149 | Human Tau-410 / 2N3R Pre-formed Fibrils Protein, His Tag | TAU-H5143 | E.coli | ||
| Alpha-Synuclein | ALN-H5114 | Human Alpha-Synuclein (A53T) Pre-formed Fibrils Protein, Tag Free | ALN-H5116 | E.coli | |
| ALN-H5115 | Human Alpha-Synuclein Pre-formed Fibrils Protein, Tag Free | ALN-H5214 | E.coli | ||
| ALN-H51H4 | Human Alpha-Synuclein Pre-formed Fibrils Protein, His Tag | ALN-H52H8 | E.coli | ||
| ALN-M51H3 | Mouse Alpha-Synuclein Pre-formed Fibrils Protein, His Tag | ALN-M52H6 | E.coli | ||
| ALN-HA1H4 | Alexa Fluor™ 647-Labeled Human Alpha-Synuclein Pre-formed Fibrils Protein, His Tag | ALN-HA1H3 | E.coli | ||
| ALN-HA114 | Alexa Fluor™ 488-Labeled Human Alpha-Synuclein Pre-formed Fibrils Protein, Tag Free | ALN-H5214 | E.coli | ||
| APP | APP-H51Ha | Human APP / Abeta42 (672-713) Pre-formed Fibrils Protein, His Tag | APP-H51H6 | E.coli | |
| APP-H51Hb | Human APP / Abeta40 (672-711) Pre-formed Fibrils Protein, His Tag | APP-H51H9 | E.coli |
More PFFs relevant products under development, stay tuned~
Pre-formed Fibrils (PFFs) Product Features
Produced by high-quality monomers: the purity and homogeneity of monomer is ≥90% as verified by MALS, more conducive to the formation of PFFs and ensure the activity.
Lower endotoxin: monomer endotoxin ≤ 1.0 EU/μg, suitable for various in vitro and in vivo experiments.
The aggregation morphology and recruitment activity of PFFs are verified by electron microscopy and ThT fluorescence assay. Free protocols are shared.
Customized products and services provided with a variety of fluorescent labeling solutions: Star Staining, Biotin labeling, chemical labeling, etc.
Stable lead time, strict control to ensure high batch-to-batch consistency, highly cost-effective;
Lower endotoxin: monomer endotoxin ≤ 1.0 EU/μg, suitable for various in vitro and in vivo experiments.
The aggregation morphology and recruitment activity of PFFs are verified by electron microscopy and ThT fluorescence assay. Free protocols are shared.
Customized products and services provided with a variety of fluorescent labeling solutions: Star Staining, Biotin labeling, chemical labeling, etc.
Stable lead time, strict control to ensure high batch-to-batch consistency, highly cost-effective;
Pre-formed Fibrils (PFFs) Product Verification Data
PFFs morphology (electron microscopy)
As shown in the figure below, the PFFs have distinct fibrous structures under electron microscopy, demonstrating that our PFFs products have accurate morphology.
TEM of Human Tau-441/2N4R Pre-formed Fibrils Protein (Cat. No. TAU-H5115).
TEM of Human Tau-441 K18 (P301L) Pre-formed Fibrils Protein (Cat. No. TAU-H5113).
PFFs Bioactivity (cell base) is consistent with the reference[3]
Tau PFFs (TAU-H5115) induced significant protein aggregation in the cytoplasm of HEK293/Human Tau-K18 (GFP) Stable Cell Line (Cat. No. CHEK-ATP087).
HEK293/Human Tau(GFP) Stable Cell Line were transduced with Human Tau-441 / 2N4R Pre-formed Fibrils Protein, Tag Free (ThT active) (Cat. No. TAU-H5115) and Human Tau-441 / 2N4R Protein, Tag Free (MALS verified) (Cat. No. TAU-H5117) respectively. The fluorescence of GFP-Tau (Green) and DAPI (Blue) were detected by confocal microscope. A. Lipo2000 transduction. B. Lipo2000 and Human Tau-441 / 2N4R Protein, Tag Free (MALS verified) transduction. C. Lipo2000 and Human Tau-441 / 2N4R Pre-formed Fibrils Protein, Tag Free (ThT active) transduction. Scale bars, 50 μm.
PFFs activity (ThT assay)
PFFs induced faster aggregation of monomer proteins than that of the control group with only monomer added.
FAQ
How does the initial monomer purity of recombinant Pre-formed Fibrils (PFFs) determine the reproducibility of seeding kinetics in neurodegenerative drug screening?
Consistent PFF performance starts with high-quality, well-characterized monomers. ACROBiosystems produces alpha-synuclein and Tau PFFs from monomers with purity and homogeneity of at least 90%, as verified by MALS. Controlling the starting material helps reduce variability caused by impurities, pre-existing aggregates, and heterogeneous protein species. PFF morphology and recruitment activity are further evaluated using electron microscopy and Thioflavin T (ThT) assays to support consistent aggregation kinetics and assay baselines.
What preparation protocols are recommended to optimize the fragmentation and endocytic internalization of Pre-formed Fibrils in primary neuronal cultures?
PFF fragment size and dispersion strongly affect cellular uptake and seeding activity. Successful cellular uptake and cell-to-cell propagation of PFF pathogenetic seeds depend on strict length distribution matrices. Large fibril aggregates may be inefficiently internalized, while excessive sonication can disrupt fibril structure and reduce seeding activity. ACROBiosystems recommends sonicating PFFs using probe sonicator or bath sonication protocols by validating via Transmission Electron Microscopy (TEM). Sonication efficiency varies with the instrument, sample volume, and application, and conditions may need to be optimized for each experimental setup. Repeated freeze-thaw cycles should be avoided.
How does specifying low endotoxin thresholds for recombinant PFF reagents de-risk in vivo efficacy studies in neurodegenerative animal models?
Endotoxin contamination can activate innate immune pathways and introduce additional inflammatory responses in neuronal cultures and animal models. Elevated endotoxin levels may promote or exacerbate microglial and astrocytic activation, creating an inflammatory background that can complicate the interpretation of PFF-induced pathology, therapeutic effects on pathological protein clearance, and neuroprotective activity. ACROBiosystems PFF products are controlled to an endotoxin level below 10 EU/mg, as determined by the LAL method / rFC method. This low-endotoxin specification helps reduce inflammatory interference in both in vitro and in vivo studies. Additional factors, including the administered dose, route of administration and appropriate experimental controls should also be considered.
Which labeling configuration is optimal for tracking PFF internalization and cell-to-cell transmission across complex 3D cerebral organoid models?
The appropriate labeling strategy depends on the experimental objective. Directly fluorescent-labeled PFFs are suitable for confocal imaging, high-content analysis, and visualization of cellular uptake and spatial distribution within 3D neural tissues. ACROBiosystems offers Alexa Fluor™ 488- and Alexa Fluor™ 647-labeled human alpha-synuclein PFFs produced by standard chemical labeling of primary amines on lysine side chains and the protein N-terminus. The two fluorophores provide flexible options for different imaging platforms and multiplexed assays. Residual unconjugated dye is removed during purification to reduce background fluorescence and the dye-to-protein molar ratio is controlled between 0.2 and 0.4.
What quality control parameters ensure long-term stability and resistance to fibril dissociation for PFF critical reagents during global assay deployment?
PFF quality control should assess starting monomer quality, protein purity, endotoxin level, fibril morphology, recruitment activity, and, where applicable, cell-based seeding activity. Electron microscopy confirms fibrillar morphology, while ThT assays evaluate recruitment activity. For applicable PFF products, unsonicated fibrils can be stored at -70°C for up to one year, whereas sonicated fibrils should not be stored for longer than eight weeks. Fibrils should not be stored on ice or at 4°C, and repeated freeze-thaw cycles should be avoided. Standardized handling, storage, and sonication procedures help reduce assay variability.
References
- 1. Stroo E, Koopman M, Nollen EA, Mata-Cabana A. Cellular Regulation of Amyloid Formation in Aging and Disease. Front Neurosci. 2017 Feb 14;11:64. doi: 10.3389/fnins.2017.00064. PMID: 28261044; PMCID: PMC5306383.
- 2. Guo JL, Lee VM. Seeding of normal Tau by pathological Tau conformers drives pathogenesis of Alzheimer-like tangles. J Biol Chem. 2011 Apr 29;286(17):15317-31. doi: 10.1074/jbc.M110.209296. Epub 2011 Mar 3. PMID: 21372138; PMCID: PMC3083182.
- 3. Sanders D W, Kaufman S K, DeVos S L, et al. Distinct tau prion strains propagate in cells and mice and define different tauopathies[J]. Neuron, 2014, 82(6): 1271-1288. doi: 10.1016/j.neuron.2014.04.047.
- Pre-formed Fibrils (PFFs) Background
- Pre-formed Fibrils (PFFs) Production and Validation
- Pre-formed Fibrils (PFFs) Product List
- Pre-formed Fibrils (PFFs) Product Features
- Pre-formed Fibrils (PFFs) Product Verification Data
- FAQ
- References