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ChromoTek Spot-Cap®

The ChromoTek Spot-Cap™ consists of an anti-Spot-tag® Nanobody (VHH), which is covalently bound to agarose. Spot-Cap is used to purify Spot-tagged proteins from cell extracts of various organisms like mammals, plants, bacteria, yeast, insects etc. Spot-Cap is engineered for gentle elution with Spot-peptide at 4°C and high yields in batch or gravity flow formats.

Specificity

Spot-Tag®

Applications

Protein purification

Conjugate

Agarose

Cat No : eca

Synonyms

Spot Affinity Gel; Spot Affinity Matrix

Validation Data Gallery

  • 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.

    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.

  • Workflow of Spot-Cap purification.

    Workflow of Spot-Cap purification.

  • 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).

    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).

  • 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.

    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.

  • 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.

    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.

  • Application of Spot-tag related products.

    Application of Spot-tag related products.

Product Information

The ChromoTek Spot-Cap™ consists of an anti-Spot-tag® Nanobody (VHH), which is covalently bound to agarose. Spot-Cap is used to purify Spot-tagged proteins from cell extracts of various organisms like mammals, plants, bacteria, yeast, insects etc. Spot-Cap is engineered for gentle elution with Spot-peptide at 4°C and high yields in batch or gravity flow formats.

DescriptionSpot-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

ApplicationsProtein purification
Specificity/TargetSpot-Tag® PDRVRAVSHWSS
Binding capacity10 mg protein (30 kDa) / 1 mL settled resin
(340 nmol / 1 mL settled resin)
ConjugateAgarose beads; bead size: ~ 90 µm (cross-linked 4 % agarose beads)
Elution buffer0.1 mM Spot-peptide or 100 mM glycine pH 2.0 at 4°C
Wash buffer compatibility1 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 purificationpH 4 - 11
Regeneration buffer100 mM glycine pH 2.0
Regeneration>5 cycles
Purification format Batch and gravity flow
Affinity (KD) Dissociation constant KD of 40 nM
RRIDAB_2827549
Storage Buffer20% ethanol
Storage ConditionShipped at ambient temperature.
Upon receipt store at 4°C. Do not freeze!
Stable for one year.
Spot-Tag® OriginEngineered 12 amino acid sequence modified from the unstructured N-terminus of beta-catenin
Cross ReactivityThe Spot-Nanobody shows minimal cross reactivity with beta-catenin.

Publications

ApplicationTitle

FEBS J

A guide to: generation and design of nanobodies.

Authors - Serge Muyldermans
View Article

J Chromatogr A

An engineered peptide tag-specific nanobody for immunoaffinity chromatography application enabling efficient product recovery at mild conditions.

Authors - Jun Ren
View Article