SLAP: Small Labeling Pair for Single‐Molecule Super‐Resolution Imaging

Abstract

Protein labeling with synthetic fluorescent probes is a key technology in chemical biology and biomedical research. A sensitive and efficient modular labeling approach (SLAP) was developed on the basis of a synthetic small-molecule recognition unit (Ni-trisNTA) and the genetically encoded minimal protein His6-10 -tag. High-density protein tracing by SLAP was demonstrated. This technique allows super-resolution fluorescence imaging and fulfills the necessary sampling criteria for single-molecule localization-based imaging techniques. It avoids masking by large probes, for example, antibodies, and supplies sensitive, precise, and robust size analysis of protein clusters (nanodomains).

Single-molecule FRET reveals the pre-initiation and initiation conformations of influenza virus promoter RNA

“…We chose a labelling approach in which fluorophore-conjugated tris- nitrilotriacetic acid ( tris NTA) (27–30) was bound to an internal His 10 tag placed at amino acid 200 within a ß-ribbon of the PB1 subunit of the RNAP (PB1 his200). Labelling was carried out in situ, resulting in a large background from unbound dye-conjugated tris NTA (Supplementary Figure S3), however a clear FRET signal could be observed.…”

Protein‐Specific, Multicolor and 3D STED Imaging in Cells with DNA‐Labeled Antibodies

“…Similar procedures could be combined with the present approach using weak‐binding DNA labels, and further increase the multiplexing capability. Further development of this concept could include genetically expressed protein tags, in particular with fluorophore labels engineered as weak binders and ultimately inside live cells . Adapting imaging parameters, such as integration time, line/frame averaging and pausing, allows the use of labels with very different exchange kinetics, hereby increasing the flexibility of the method.…”

Multivalent Chelators for In Vivo Protein Labeling

“…[41] Taking advantage of bright, photostable organic dyes conjugated to trisNTA, different intracellular POIs were efficiently decorated with trisNTAfluorophore constructs for confocal laser scanning microscopy (CLSM) and direct stochastic optical reconstruction microscopy (dSTORM) in various cell types.T he cytoskeletal protein b-actin (Figure 3a), the nuclear envelope protein lamin A, and the transporter associated with antigen process-ing (TAP) in the endoplasmic reticulum (ER) membrane were visualized at 40 nm resolution. [41] Taking advantage of bright, photostable organic dyes conjugated to trisNTA, different intracellular POIs were efficiently decorated with trisNTAfluorophore constructs for confocal laser scanning microscopy (CLSM) and direct stochastic optical reconstruction microscopy (dSTORM) in various cell types.T he cytoskeletal protein b-actin (Figure 3a), the nuclear envelope protein lamin A, and the transporter associated with antigen process-ing (TAP) in the endoplasmic reticulum (ER) membrane were visualized at 40 nm resolution.…”

“…Interestingly,b yc omparing conventional immunostaining (primary-and secondary-antibody (AB) labeling) with trisN-TA targeting,aseverely increased cluster dimension for immunostained TAPw as determined (Figure 3c;A Bs:7 1AE 8nmv s. trisNTA: 49 AE 7). [41].C opyright Wiley-VCH,2 015. d) Four different single imaging rounds of acell transfected with His 10 -tagged lamin Aobtained by single-epitope repetitive imaging (SERI). This showed that the trisNTA- Figure 3.…”