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You may have noticed the recent publication “A peptide tag-specific nanobody enables high-quality labeling for STORM imaging” of Virant et al (2018) in Nature Communications doi: 10.1038/s41467-018-03191-2, where for the first time a peptide-tag specific Nanobody was applied in dSTORM imaging: The authors have described and discussed the performance of a BC2 peptide-tag specific Nanobody to image BC2 peptide tag fusion proteins in super resolution microscopy, i.e. dSTORM. This Blog features that BC2 peptide-tag specific Nanobody, which ChromoTek markets under the names Spot-Label® and Spot-Trap®. In addition, this Blog figures the significantly improved version of the BC2 peptide -tag, which is called Spot-Tag®.

 

Spot-Label and Spot-Trap are conjugates of the BC2-tag Nanobody

The monovalent and bivalent derivatives of the BC2 peptide-tag Nanobody, which are discussed in the publication Virant et al (2018), are available from ChromoTek. The Spot-Tag Nanobody is marketed using the product names Spot-Label, when conjugated to a fluorophore, or Spot-Trap, when conjugated to a solid matrix.


Spot-Tag
is a significantly improved version of the BC2 peptide-tag

The affinity of the referenced Nanobody respectively Spot-Label/Spot-Trap to the advanced Spot-Tag peptide is significantly higher than to BC2 peptide-tag and therefore further reduces background.


What are the differences between BC2-Tag and Spot-Tag?


Four of the 12 amino acids from BC2 peptide-tag (green, top) have been replaced by rational design to form the Spot-Tag peptide (blue, bottom):

BC2-Tag - Spot-Tag 1801.png

As a result, the Spot-Tag Nanobody has a higher affinity to Spot-Tag than to the BC2 peptide-tag.

Why should I use Spot-Tag instead of BC2-tag?

Because the Spot-Tag peptide is preferentially bound with low nanomolar affinity by the Spot-Tag nanobody.

 

Where can I obtain Spot-Tag peptide expression vectors?

You can receive Spot-Tag peptide expressions vectors free of charge from ChromoTek here: https://resources.chromotek.com/spot-tag


 

Where can I buy monovalent (mono)BC2-Nb and bivalent bivBC2-Nb Nanobody in Virant et al?
What are corresponding ChromoTek product names that are referenced in Virant et al (2018)?

ChromoTek sells fluorophore conjugated Nanobodies under its product name Spot-Label. See below and/or send email to info@chromotek.com

Virant et al abbreviation

Virant et al name

Corresponding ChromoTek
product name & code

ChromoTek tested Application

BC2-Nb or monoBC2-Nb

Monovalent anti-BC2-Nanobody

Spot-Label, monovalent:

etx-50 (test size) and etx-250 & anti-alpaca secondary: PABAL1-10 (test size)
 Western Blot 

monoBC2-NbAF647 (NHS)

Monovalent anti-BC2-Nanobody, NHS conjugated to Alexa647

Spot-Label_Atto488, for Western blot (WB), monovalent: ema488-10 (test size)

 

 Western Blot

bivBC2- NbAF647 (sort)

Bivalent anti-BC2-Nanobody, sortase conjugated to Alexa647 Product not launched yet. Stay tuned  

bivBC2- NbAF647 (NHS)

Bivalent anti-BC2-Nanobody, NHS conjugated to Alexa647 Spot-Label_Atto594 for Immunofluorescence (IF), bivalent: eba594-10  

IF,

STED

More Spot-Label conjugates with additional fluorophores will be launched. Please inquire for updates (support@chromotek.com).


Does BC2-tag or Spot-Tag create background in immunofluorescence microscopy?

No – Background is caused by the Nanobody binding non-specifically to other proteins or peptides. Spot-Tag is a peptide that binds with an up to 10-times higher affinity to the Nanobody than the BC2-tag.

As the anti-BC2 Nanobody was originally generated against beta-catenin, Virant et al (2018) have assessed the background staining of endogenous beta-catenin. The authors conclude that the BC2-epitope of beta-catenin has only a minor impact on imaging BC2-tagged proteins. Due to its superior binding to the Spot-Tag, the Spot-Tag Nanobody causes even less beta-catenin-derived background staining when working with Spot-tagged proteins. This is strongly supported by ChromoTek’s imaging data, in which no background from endogenous beta-catenin was observed.

 

How is Spot-Tag characterized?

  • 12 amino acid sequence: PDRVRAVSHWSS
  • Size: 1.4 kDa
  • pI: 10.4
  • Extinction coefficient: 5,500 L·mol−1cm−1
  • Expression tested in bacteria, yeast, mammalian cell lines and insect cells; other cells/organisms not tested
  • Shown to work both as N-terminal and C-terminal fusion
  • Biological function of the Spot-tagged fusion protein is not impaired
  • High affinity, dissociation constant in low nanomolar range

 

Where do I find more information about the Spot-Nanobody?

See https://www.chromotek.com.

 

Can Spot-Tag and Spot-Tag Nanobody be used for STED, too?


Yes, Spot-Tag and its Nanobody can be also used for STED SRM technologies. Below see two STED images using Spot-Label594, i.e. anti-Spot-Tag Nanobody, bivalent, NHS conjugated to Atto594: HeLa cells expressing Vimentin-Spot-Tag (left) and HeLa cells expressing Actin-Chromobody-Spot-Tag (right) were immunostained with anti-Spot-Label594 (1:1000). Gated STED images were acquired with a Leica TCS SP8 STED 3X microscope with pulsed White Light Laser excitation at 590 nm and pulsed depletion with a 775 nm laser. Objective: 100x Oil STED White, NA: 1.4. Pixel size: 21 x 21 nm; z-Step size of z-Stacks: 0.16 µm. Images were deconvolved with Huygens Professional (SVI). STED images were recorded at the Core Facility Bioimaging at the Biomedical Center, LMU Munich.

     
Spot-Label Blog 1801-1.png  Spot-Label Blog 1801-2.png

 

Why are Nanobodies particularly well suited for imaging applications?

The anti-Spot Nanobody conjugated to a fluorophore is called Spot-Label and has the smallest epitope label displacement, also called linkage error, of all antibodies when labelling a target. This is of particular importance for SRM imaging. In addition, Spot-Label has a superior tissue and cell penetration rate compared to IgGs.
Alpaca_heavy chain ab_Nano-Booster.png


Is Spot-Tag validated for other applications than IF? – What applications can Spot-Tag be used for?

Yes, Spot-Tag in combination with Spot-Trap or Spot-Label can also be used for:

  • Immunoprecipitation
  • Affinity protein purification (native and non-native elution conditions)
  • Immunofluorescence & imaging (wide field, confocal, dSTORM, STED)
  • Western blotting

What Literature has already been published?

Braun, M. B. et al.  Peptides in headlock – a novel high-affinity and versatile peptide binding nanobody for proteomics and microscopy, Sci. Rep. 6, 19211; doi: 10.1038/srep19211 (2016).

Bruce, V. J. and McNaughton, B. R.  Evaluation of Nanobody Conjugates and Protein Fusions as Bioanalytical Reagents, Anal. Chem. doi: 10.1021/acs.analchem.7b00470 (2017)

Virant, D., Traenkle, B., Maier, J., Kaiser, P. D., Bodenhoefer, M., Schmees, D., Vojnovic, I., Pisak-Lukáts, B., Endesfelder, U. and Rothbauer, U. 2018. A peptide tag-specific nanobody enables high-quality labeling for STORM imaging, Nature communications; doi: 10.1038/s41467-018-03191-2 (2018)

Topics: Spot-Tag, Spot-Trap, Spot-Label, Capture/detection System, Spot Nanobody, Nanobody, Spot immunofluorescence, Spot super resolution microscopy, Spot STED, BC2tag

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