anti-Spot-Tag VHH/ Nanobody conjugated to fluorescent dye for immunofluorescence, microscopy, and immunoblotting of Spot-tagged proteins
Spot-Label binds to the Spot-Tag sequence PDRVRAVSHWSS at the N-terminus or the C-terminus.
- Immunofluorescence (IF)
- Wide-field fluorescence and confocal microscopy, super Resolution Microscopy (SRM)
- Western blotting
|Product Spot-Label ATTO488||Size 10 µL||Code eba488-10||Price $ 100||Buy +|
|Product Spot-Label ATTO488||Size 50 µL||Code eba488-50||Price $ 395||Buy +|
|Product Spot-Label ATTO594||Size 10 µL||Code eba594-10||Price $ 100||Buy +|
|Product Spot-Label ATTO594||Size 50 µL||Code eba594-50||Price $ 395||Buy +|
Coupled Nanobody/ VHH
Recombinant, monoclonal bivalent anti-Spot single domain antibody (sdAb) fragment
Spot-Tag sequence PDRVRAVSHWSS
Alpaca/recombinant VHH domain, monoclonal
IF: 1:10,000; Immunoblotting: 1:1,000 to 1:20,000 Optimal working concentration is application-dependent and should be determined by testing
Wide-field or epifluorescence microscopy; confocal microscopy; super-resolution microscopy e.g. STED, dSTORM
Purified recombinant protein in PBS supplemented with preservative 0.09 % sodium azide
10 μL, 50 μL
Because of the small size of the VHH these volumes correspond to about 10 times the volume of conventional IgG antibodies.
0.5 – 1 mg/mL (conjugates)
Shipped at ambient temperature. Upon receipt store at 4°C. Do not freeze. Protect from light.
Validated in cell culture & cell lines and fixed cells
Dissociation constant KD of 6 nM
Plus avidity effect of about 400 times from bivalent Spot-Label format
Which Nano-Booster and Nano-Label conjugates are recommended for super-resolution microscopy?
Nano-Boosters and Nano-Labels are highly suitable for Super-Resolution Microscopy. Due to their small size (2-3 nm), they minimize the linkage error and provide a more precise and dense staining, than conventional antibodies (15 nm linear dimension. The selection of a Nano-Booster and Nano-Label conjugate depends on your microscope setup and lasers. We recommend for:
- STED: ATTO647N, Abberior STAR 635P
- STORM: Alexa Fluor 647, ATTO488
- SIM: ATTO488/594
Are Nano-Booster and Nano-Labels applicable for live-cell imaging?
Yes, if the fusion-tag is on the cell surface.
Nano-Boosters and Nano-Labels are small proteins and therefore don’t penetrate through non-permeabilized cell membranes. Hence, if your fusion-protein is intracellular, you may want to apply protein transduction methods (e.g. electroporation) or reagents, however from our experience, the most efficient way is to microinject the Nano-Boosters and Nano-Labels.
Can I do a simultaneous co-staining with two or more Nano-Boosters and Nano-Labels?
Yes, you can combine the Nano-Boosters and Nano-Labels. For example, if you typically use the Nano-Boosters in a 1:200 dilution, you should add 1 µL each of gba488 and rba594 to 200 µL of blocking solution for a co-staining.
How many dye molecules are coupled to Nano-Boosters and Nano-Labels?
Each Nano-Booster and Nano-Label molecule carries on average 1-2 fluorophores.
Note: Nano-Boosters labeled with Atto647N carry a maximum of 1 fluorophore per VHH.
What is the protocol for live-cell Nano-Booster and Nano-Label staining of the extracellular fusion protein?
Incubate the cells with 1:25 Nano-Booster or Nano-Label in growth media for 15 min at +4°C, wash and image. This protocol will highlight just the plasma membrane pool of your fusion protein.
Do Nano-Boosters and Nano-Labels penetrate though the cell membranes of live cells?
No. Nano-Boosters and Nano-Labels are small proteins and therefore don’t penetrate through non-permeabilized cell membranes. If you need to deliver Nano-Booster and Nano-Labels into live cells, you may want to apply protein transduction methods (e.g. electroporation) or reagents, however from our experience, the most efficient way is to microinject the Nano-Boosters and Nano-Labels.
Is it possible to conjugate Nano-Boosters and Nano-Labels to other fluorophores?
Yes. You can label the ChromoTek GFP-Binding Protein (GFP VHH, product code: gt-250), RFP-Binding Protein (RFP VHH, product code: rt-250) and Spot-Binding Protein (Spot VHH, product code: etx-250) with NHS-activated fluorescent dyes following the instructions of the dye manufacturer.
Note: Spot VHH contains a sortase-tag at its C terminus (sortase recognition motif LPETG) which can be used for conjugation.
First peptide-tag/ Nanobody system used in Super Resolution Microscopy
Virant et al. report in “A peptide tag-specific nanobody enables high-quality labeling for STORM imaging”, Nature Communications (2018), doi: 10.1038/s41467-018-03191-2, the first peptide-tag specific Nanobody that was applied in dSTORM imaging: BC2 peptide-tag specific Nanobody to image BC2 peptide-tagged proteins. ChromoTek markets that BC2 peptide-tag specific Nanobody under the product names Spot-Label® and Spot-Trap®.
- Capture and detection tag without compromises in applications
- Higher image resolution and higher labelling density
- Superior accessibility and labelling of epitopes in crowded cellular/organelle environments
- Strong avidity effect from bivalent form of Spot-VHH
- Fulfills highest requirements on antibody validation: structure and function characterized
Spot-Label samples available
Only for research applications, not for diagnostic or therapeutic use!