Source 1primary paper2010ChemBioChem
Toolkit/β-tubulin-SNAP
β-tubulin-SNAP
Also known as: β-tubulin–SNAP fusion protein
Taxonomy: Mechanism Branch / Component. Workflows sit above the mechanism and technique branches rather than replacing them.
Summary
Towards this end, we expressed β-tubulin as a C-terminal fusion of SNAP-tag (β-tubulin-SNAP) in U2OS cells.
Usefulness & Problems
No literature-backed usefulness or problem-fit explainer has been materialized for this record yet.
Published Workflows
Objective: Develop and apply a targeted photoswitchable SNAP-tag substrate for specific labeling of cellular proteins and STORM nanoscopy of microtubules.
Why it works: The workflow couples a benzylguanine-targeted Cy3-Cy5 photoswitch to SNAP-tag fusion proteins, allowing specific covalent labeling of the structure of interest and then STORM reconstruction from single-emitter localization. The paper argues that the small size and stoichiometric labeling of SNAP-tag help avoid problems associated with antibody-based targeting.
benzylguanine-mediated targeting of a Cy3-Cy5 photoswitch to SNAP-tag fusion proteinssingle-emitter localization through repeated photoswitching for STORM reconstructionchemical probe synthesisSNAP-tag fusion protein labelingconfocal specificity checkSTORM imaging
Stages
- 1.Probe synthesis and in vitro SNAP reactivity check(library_build)
This stage establishes that the synthesized BG-Cy3-Cy5 probe can be produced and can react with SNAP-tag before cellular imaging experiments.
Selection: Obtain a photoswitchable benzylguanine probe that reacts with SNAP-tag
- 2.Cellular labeling specificity check(confirmatory_validation)
The authors use confocal colocalization with α-tubulin immunostaining to confirm that the chemical labeling marks the intended structure before super-resolution reconstruction.
Selection: Verify that BG-Cy3-Cy5 specifically labels β-tubulin-SNAP in fixed cells and localizes to microtubules
- 3.STORM imaging and reconstruction of labeled microtubules(confirmatory_validation)
This stage tests whether the targeted photoswitchable labeling strategy supports STORM reconstruction of microtubules below the diffraction limit.
Selection: Demonstrate nanoscale imaging performance on the labeled structure
Steps
- 1.Synthesize BG-Cy3-Cy5 from commercially available materialsengineered probe
Create a benzylguanine-linked Cy3-Cy5 photoswitchable substrate for SNAP-tag targeting
The probe must exist before its SNAP reactivity and cellular imaging utility can be tested.
- 2.Test in vitro reaction of BG-Cy3-Cy5 with SNAP-tagprobe and target tag
Verify that the synthesized probe reacts with SNAP-tag before cell-based experiments
In vitro reactivity is a lower-complexity prerequisite for interpreting later cellular labeling results.
- 3.Express β-tubulin-SNAP in U2OS cellscellular target construct
Place SNAP-tag on microtubules for targeted labeling
A SNAP-tag fusion target is required before fixed-cell labeling with BG-Cy3-Cy5 can be performed.
- 4.Fix cells and incubate with BG-Cy3-Cy5 followed by washinglabeling probe and target construct
Chemically label β-tubulin-SNAP in fixed cells
The paper emphasizes that post-fixation labeling is a prerequisite for compatibility with STORM.
- 5.Perform α-tubulin immunostaining control and confocal colocalization imaging
Confirm correct localization and specificity of SNAP-directed labeling before STORM
This control reduces the risk that later super-resolution structures reflect nonspecific labeling rather than microtubules.
- 6.Image labeled samples under STORM conditions and acquire 5000 frames
Collect photoswitching image data for single-molecule localization reconstruction
STORM acquisition follows confirmation of specific labeling so that the reconstruction can be interpreted as the target structure.
- 7.Reconstruct and filter single-molecule localization images
Generate super-resolved images and remove nonstructured background
Image reconstruction and filtering are required after acquisition to resolve nanoscale microtubule structure from the raw frame series.
Taxonomy & Function
Primary hierarchy
Mechanism Branch
Component: A low-level protein part used inside a larger architecture that realizes a mechanism.
Techniques
No technique tags yet.
Target processes
localizationInput: Light
Validation
Cell-freeBacteriaMammalianMouseHumanTherapeuticIndep. Replication
Observations
successMammalian Cell Lineapplication demohumanU2OS
confocal fluorescence imaging
Inferred from claim c3 during normalization. BG-Cy3-Cy5 specifically labels β-tubulin-SNAP in fixed U2OS cells, as indicated by colocalization with α-tubulin immunostaining. Derived from claim c3. Quoted text: Confocal fluorescence imaging demonstrates a highly specific labeling of β-tubulin-SNAP with Cy3-Cy5, as shown by the colocalization of the signals from the immunostaining of α-tubulin and the SNAP-tag labeling.
Source:
Supporting Sources
Ranked Claims
SNAP-tag labeling addresses limitations of antibody-based STORM labeling by enabling specific, fast, stoichiometric, quantitative, and post-fixation-compatible labeling with a smaller tag.
The SNAP-tag is a small and highly soluble protein of 20 kD... The labeling is highly specific, fast, stoichiometric and quantitative. Furthermore, SNAP-tag labeling can be achieved after fixation of cells; this is a prerequisite for its compatibility with STORM.
Microtubules measured by this SNAP-tag STORM method were smaller than microtubules measured with antibody-based STORM.
The characteristic dimensions of these structures measured by this method are 40±10 nm in diameter... It is noteworthy that this measured size of the microtubule is smaller than that measured with STORM based on antibody staining (60 nm).
antibody based STORM measurement 60 nmmeasured microtubule diameter 40±10 nm
STORM imaging with the described SNAP-targeted photoswitchable probe revealed SNAP-tagged microtubule structures with about 25 nm resolution.
Stochastic Optical Reconstruction Microscopy (STORM) reveals SNAP-tagged microtubule structures with ∼25 nm resolution.
resolution 25 nm
BG-Cy3-Cy5 specifically labels β-tubulin-SNAP in fixed U2OS cells, as indicated by colocalization with α-tubulin immunostaining.
Confocal fluorescence imaging demonstrates a highly specific labeling of β-tubulin-SNAP with Cy3-Cy5, as shown by the colocalization of the signals from the immunostaining of α-tubulin and the SNAP-tag labeling.
The paper introduces a photoswitchable O6-benzylguanine derivative for super-resolution microscopy of SNAP-tagged proteins.
We introduce a photoswitchable O6-benzylguanine derivative and demonstrate its use for super-resolution microscopy of SNAP-tagged proteins based on single fluorophore localization.
Approval Evidence
1 source3 linked approval claimsfirst-pass slug tubulin-snap
Towards this end, we expressed β-tubulin as a C-terminal fusion of SNAP-tag (β-tubulin-SNAP) in U2OS cells.
Source:
comparative measurementsupports
Microtubules measured by this SNAP-tag STORM method were smaller than microtubules measured with antibody-based STORM.
The characteristic dimensions of these structures measured by this method are 40±10 nm in diameter... It is noteworthy that this measured size of the microtubule is smaller than that measured with STORM based on antibody staining (60 nm).
Source:
performancesupports
STORM imaging with the described SNAP-targeted photoswitchable probe revealed SNAP-tagged microtubule structures with about 25 nm resolution.
Stochastic Optical Reconstruction Microscopy (STORM) reveals SNAP-tagged microtubule structures with ∼25 nm resolution.
Source:
specificitysupports
BG-Cy3-Cy5 specifically labels β-tubulin-SNAP in fixed U2OS cells, as indicated by colocalization with α-tubulin immunostaining.
Confocal fluorescence imaging demonstrates a highly specific labeling of β-tubulin-SNAP with Cy3-Cy5, as shown by the colocalization of the signals from the immunostaining of α-tubulin and the SNAP-tag labeling.
Source:
Comparisons
No literature-backed comparison notes have been materialized for this record yet.
Ranked Citations
- 1.