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: etb-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
- 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
“The images are really nice, and we feel that they demonstrate how well the Spot-Tag system works.”
Professor, Stockholm University, Sweden
“I have been using Spot Nanobody (Spot-Label 594) for labeling fixed cells and am very happy with the product.“
Instructor, La Jolla Inst. for Immunology, CA, USA
“They are going great! We expect to submit our first paper using Spot technology within the next month!”
Associate Professor, The College of William & Mary, Williamsburg, VA, USA
Spot-Label samples available
Only for research applications, not for diagnostic or therapeutic use!