I purified tau phf from cells, but found that purified tau phf tends to form clumps in negative stain em images. how to improve to get separated filaments ?
To improve the purification and preparation of Tau paired helical filaments (PHF) for negative stain electron microscopy (EM), focus on fragment selection, buffer optimization, and the addition of surfactants to reduce clumping.
Optimization of Tau Filament Assembly and Selection
Using specific truncated Tau fragments can yield filaments that more closely resemble the stable core found in Alzheimer's disease (AD) without requiring external cofactors that may cause heterogeneity.
* Utilize the Tau (297–391) Fragment: The dGAE fragment (residues 297–391) encompasses the proteolytically stable core of PHFs. Unlike full-length Tau, dGAE can self-assemble spontaneously without anionic cofactors like heparin, which often produce structurally heterogeneous filaments (Direct, High; PMID: 31721178).
* Controlled Assembly Conditions: Optimal assembly of dGAE PHFs occurs when 300 µM of protein is incubated in 10 mM phosphate buffer (pH 7.4) with 10 mM DTT at 37°C. Continuous agitation (e.g., 400 oscillations per minute) for 48 hours is necessary to ensure consistent filament formation (Direct, High; PMID: 31721178).
* Reducing Agents: The presence of DTT during incubation is critical for maintaining the assembly environment (Direct, High; PMID: 31721178).
Strategies to Prevent Filament Clumping
Aggregation and clumping are common failure modes in filament preparation for EM. Several methodological adjustments can improve dispersion:
* Addition of Detergents or Surfactants: For samples exhibiting bad aggregation or clumping, adding detergents or other surfactants is highly recommended. This is a primary optimization method for filamentous assemblies (Direct, Medium; PMID: 31553060).
* Buffer and Ionic Strength: Small adjustments to the pH and ionic strength of the sample buffer can significantly impact particle dispersion, although this must be determined empirically for each specific protein complex (Direct, Medium; PMID: 31553060).
Grid Preparation for Improved Dispersion
The physical preparation of the EM grid is as vital as the biochemistry of the sample for obtaining separated filaments:
* Wash Steps: After applying the filament sample (e.g., 4 µL) to the grid and blotting, washing with filtered Milli-Q water is essential to remove salts and residual buffer components that can interfere with negative stain clarity (Direct, High; PMID: 31721178).
* Modified Application Techniques: If particle density is too low in certain areas, applying the sample two or more times to the same grid (with washes in between) can increase the number of visible filaments (Direct, Medium; PMID: 31553060).
* Support Media Alternatives: If standard carbon films fail to provide good dispersion, using graphene oxide as a support film or utilizing holey-gold grids may provide better results (Direct, Medium; PMID: 31553060).
Negative Stain Protocol for Tau PHF
Consistent staining is required to visualize the characteristic 72.5 nm crossover repeat and 9.5–17.5 nm width of PHFs:
* Detection Verification: To confirm the filaments are indeed Tau PHFs, immunogold labeling with antibodies like mAb 423 (specific for Tau truncated at Glu-391) or T22 can be performed directly on the grids before negative staining (Direct, High; PMID: 31721178).
Unverified Citations
The following sources failed to support their assigned claims after 3 verification rounds designed to ensure only high-confidence, relevant references are retained:
- PMID:31553060 — 1% glutaraldehyde is typically used before washing and resin embedding
Failed: conclusion — The paper mentions glutaraldehyde as a crosslinking agent but does not specify a 1% concentration or its use specifically before resin embedding. - PMID:31721178 — 1% glutaraldehyde is typically used before washing and resin embedding
Failed: conclusion — The paper uses 0.1% glutaraldehyde, whereas the claim specifies 1% glutaraldehyde. - PMID:31721178 — Adsorption can be improved by treating these films with glow discharge to make them more hydrophilic
Failed: mechanism,conclusion — The cited paper does not mention glow discharge or its use to improve adsorption. - PMID:31721178 — 22-µm) and allowed to sit on the grid for 1 minute before blotting and air-drying
Failed: conclusion — The claim specifies a 22-µm concentration/size which is not supported by the paper; the paper uses 2% w/v uranyl acetate filtered through a 0.22-µm filter.