Spot Capture and Detection System

ChromoTek’s Spot-Tag® is the first peptide tag, which is bound by a Nanobody, and which is universal applicable in protein capture and detection applications. The Spot-Tag is an inert 12 amino acid peptide-tag (PDRVRAVSHWSS), which is specifically bound by the highly stable and robust anti-Spot-Nanobody.

CRISPR/Cas

Undoubtedly, affinity tags are valuable tools for studying heterologously overexpressed proteins. However, tag sequences can also be introduced to endogenous genes, which are expressed under their own promoters. CRISPR/Cas9 is a popular and powerful tool for the generation of such constructs. By introducing the Spot-Tag using the CRISPR/Cas9 technology, proteins can be studied at the endogenous expression level. Using both Spot-Trap® and Spot-Label®, endogenous Spot-tagged protein from a CRISPR/Cas9-engineered cell line was used for immunoprecipitation and Western blotting as well as for immunofluorescence applications.

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ELISA

The anti-Spot Nanobody allows detection and capture of Spot-tagged proteins of interest in various ELISA setups.

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Immunofluorescence

Spot-tagged proteins can be detected by immunofluorescence using various microscopy techniques, including epi-fluorescence, confocal, and super resolution microscopy. ChromoTek’s Spot-Label® offers a choice of different fluorophores for detection. Spot-Label is the first Nanobody used in super resolution microscopy for the detection of a peptide tag. Because of its small size, Spot-Label has a minimal epitope-to-label displacement, which leads to better resolution. In addition, the small size allows excellent tissue penetration.

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Immunoprecipitation

Spot-tagged proteins can be immunoprecipitated using Spot-Trap. The Spot-Tag can be placed either at the N-terminus or at the C-terminus of the protein of interest. Spot-Trap is available both as agarose beads or as magnetic agarose beads and is characterized by a remarkably low unspecific background binding.

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Mass spectrometry

Interactome studies can be performed using Spot-Trap for co-immunoprecipitations followed by mass spectrometric analysis. Trypsinization (i.e. the proteolytic digest of the protein sample with trypsin) can be done directly on the beads, without elution of the immunoprecipitated proteins. Due to the small size of the anti-Spot-Nanobody, only four to five defined Nanobody-derived peptides are generated during the on-bead trypsinization.

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Protein purification

Spot-tagged proteins can be affinity purified using Spot-Cap (i.e. a dedicated anti-Spot Nanobody covalently coupled to magnetic agarose). This purification comprises (i) cell lysis, (ii) cell centrifugation to remove cell debris, (iii) binding of the Spot-tagged protein to Spot-Cap, (iv) washing to remove unwanted host cell proteins, and finally (v) elution by Spot-peptide or pH shift. High yield and purity protein is typically obtained. Spot-Cap is an economic purification matrix, because it has a high binding capacity and can be regenerated frequently.

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Western blotting

The anti-Spot VHH can be used in Western blotting for the detection of Spot-tagged fusion proteins. With a suitable fluorescent imaging system, Spot-Label can be directly applied to the Western blot membrane, and no secondary antibody is needed. Alternatively, the anti-Spot Nanobody can be detected with a labelled anti-llama secondary antibody.

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Looking for references? Please check our literature database.

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