ChromoTek Spot-Cap® and Peptide

Validation Data Gallery

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  Purification of Spot-tagged GFP with Spot-Cap from HEK293T cell lysate in gravity flow column format. Elution 1-6: 6 fractions à 2 CVs Spot-peptide (100 μM) in PBS.

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  Workflow of Spot-Cap purification.

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  Spot-Cap has a higher selectivity than anti-DYKDDDDK resin. While Spot-Cap is optimized for minimal contamination of host cell proteins, the anti-DYKDDDDK resin binds significant amounts of mammalian proteins. Incubation of control HEK293T cell lysate expressing no tagged protein with Spot-Cap or anti-DYKDDDDK resin. Elution with 100 μM Spot-peptide or 0.1 g/L DYKDDDDK-peptide. Similar results were obtained for yeast, bacteria, and insect cell lysates (data not shown).

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  The elution efficiency of Spot-Cap is constant even after 4 regeneration cycles. Elution of Spot-tagged PCNA with Spot-peptide. R0: Reference (elution fraction of Spot-PCNA without regeneration); R1-R4: Elution fraction of Spot-PCNA after 1, 2, 3, and 4 regeneration cycles with 100 mM glycine pH 2.0.

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  Purification of Spot-tagged GFP with Spot-Cap from HEK293T cell lysate in batch format. Elution: 6 fractions à 2 BVs Spot-peptide (100 μM) in PBS.

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  Application of Spot-tag related products.

Spot-Cap® is a Nanobody-based affinity resin optimized for one-step protein purification of Spot-tagged proteins.

Cat No. eca-ep

Specificity

Spot-Tag^®

Applications

Protein purification

Conjugate

Agarose

Synonyms

Spot Affinity Gel; Spot Affinity Matrix

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Product Information

Spot-Cap® is a Nanobody-based affinity resin optimized for one-step protein purification of Spot-tagged proteins.

• Description: Spot-Cap® is a Nanobody-based affinity resin optimized for one-step protein purification of Spot-tagged proteins:

  • Economic: high binding capacity and multiple regeneration cycles

  • Fast and gentle one-step purification at 4°C preserves protein stability and function

  • Tight and highly selective binding of a single repeat Spot-Tag

  • High specificity leads to minimal contamination of host cell proteins

  • No contamination of antibody heavy and light chains

• Applications: Protein purification
• Specificity/Target: Spot-Tag^® PDRVRAVSHWSS
• Binding capacity: 10 mg protein (30 kDa) / 1 mL settled resin ; (340 nmol / 1 mL settled resin)
• Conjugate: Agarose beads; bead size: ~ 90 µm (cross-linked 4 % agarose beads)
• Elution buffer: 0.1 mM Spot-peptide or 100 mM glycine pH 2.0 at 4°C
• Wash buffer compatibility: 1 M NaCl, 2 M Urea, 10 mM DTT, 10 mM ß-mercaptoethanol, 10 mM TCEP, 2% DDM, 2% NP-40, 2% Triton X-100
• pH range for purification: pH 4 - 11
• Regeneration buffer: 100 mM glycine pH 2.0
• Regeneration: >5 cycles
• Purification format: Batch and gravity flow
• Affinity (KD): Dissociation constant K_D of 40 nM
• RRID: AB_2827549
• Storage Buffer: 20% ethanol
• Storage Condition: Shipped at ambient temperature. ; Upon receipt store at 4°C. Do not freeze! ; Stable for one year.
• Spot-Tag® Origin: Engineered 12 amino acid sequence modified from the unstructured N-terminus of beta-catenin
• Cross Reactivity: The Spot-Nanobody shows minimal cross reactivity with beta-catenin.

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