Spot-Label

Description
anti-Spot-Tag VHH/ Nanobody conjugated to fluorescent dye for immunofluorescence, microscopy, and immunoblotting of Spot-tagged proteins

Conjugates
ATTO488
ATTO594
Unconjugated

Specificity
Spot-Label binds to the Spot-Tag sequence PDRVRAVSHWSS at the N-terminus or the C-terminus.

Applications

  • Immunofluorescence (IF)
  • Wide-field fluorescence and confocal microscopy, super Resolution Microscopy (SRM)
  • Western blotting
Product Size Code Price Buy
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

Specificity
Spot-Tag sequence PDRVRAVSHWSS

Host/isotype
Alpaca/recombinant VHH domain, monoclonal

Available conjugates
ATTO488, ATTO594

Recommended dilution
IF: 1:10,000; Immunoblotting: 1:1,000 to 1:20,000 Optimal working concentration is application-dependent and should be determined by testing

Microscopy techniques
Wide-field or epifluorescence microscopy; confocal microscopy; super-resolution microscopy e.g. STED, dSTORM

Form
Purified recombinant protein in PBS supplemented with preservative 0.09 % sodium azide

Size
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.

Protein concentration
0.5 – 1 mg/mL (conjugates)

Storage instructions
Shipped at ambient temperature. Upon receipt store at 4°C. Do not freeze. Protect from light.

Validation
Validated in cell culture & cell lines and fixed cells

Affinity
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®.

Benefits

  • 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

Request a free sample here and test the all-rounder Spot in you experiment.

Download Whitepaper
Order Free Test-Sample
Email newsletter

Only for research applications, not for diagnostic or therapeutic use!