Spot-Cap

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.

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.

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

Specificity
Spot-Tag sequence PDRVRAVSHWSS at the N-terminus or the C-terminus of the fusion protein

Applications
Protein purification
For immunoprecipitation (IP), Co-IP, Mass spectrometry use Spot-Trap

Properties
One-step protein purification
High binding capacity
Multiple regeneration cycles
Effective elution at 4°C with Spot-peptide or pH shift

Synonyms
Spot Affinity Gel
Spot Affinity Matrix

Product	Size	Code	Price	Buy
Product Spot-Cap™	Size 2 mL	Code eca-2	Price $ 1690	Buy +
Product Spot-Cap™ and peptide	Size 0.1 mL; 1 mg	Code eca-ep	Price $ 170	Buy +
Product Spot-peptide	Size 1 mg	Code ep-1	Price $ 85	Buy +
Product Spot-peptide	Size 10 mg	Code ep-10	Price $ 450	Buy +

Product type
anti-Spot-Tag Nanobody (V_HH) conjugated to agarose beads

Binding capacity
10 mg protein (30 kDa) / 1 mL settled resin (340 nmol / 1 mL settled resin)

Specificity
Spot-Tag sequence motif PDRVRAVSHWSS

Elution conditions
0.1 mM Spot-peptide or 100 mM glycine pH 2.0 at 4°C

Regeneration buffer
100 mM glycine pH 2.0

Regeneration
>5 cycles

Purification format
Batch and gravity flow

Form
50% slurry

Matrix, particle size
4% agarose, 90 µm

pH range for purification
pH 4 - 11

Chemical stability
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

RRID
AB_2827549

Storage instructions
Shipped at ambient temperature. Upon receipt store at 4°C. Stable for one year.

Protocol
Protocol Spot-Cap (PDF)

Datasheet
Spot peptide (PDF)

Brochure
Spot-Cap product brochure (PDF)

SDS
SDS Spot-Cap (PDF)

SDS Spot peptide (PDF)

Benefits

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

Economic

One-step purification with high purity and yield reduces costs and efforts
Constant high purification results over >5 regeneration cycles
High binding capacity and high elution efficiency
Effective elution at acidic conditions or with Spot-peptide in a concentration of just 0.1 mM. Other purification resins require high peptide concentration of up to 2 mM for elution.

Versatile

For any kind of protein, especially low expressed or sensitive proteins, membrane or secreted proteins, metalloproteins and multiple subunit protein complexes.
Broad buffer compatibility and inert tag allow easy integration in experimental workflows
Suitable for all commonly used expression hosts, e.g. bacteria, yeast, insect cells, and mammalian cells
Engineered for optimal elution efficiency and high yield at 4°C in batch or gravity flow formats

Spot-Tag: Inert 12 amino acid peptide tag

Sequence: PDRVRAVSHWSS (1.4 kDa)
Short, flexible Spot-Tag preserves folding and function of fusion protein
N- and C-terminal fusion for flexibility in cloning and protein expression
No tag removal needed after purification because Spot-Tag is compatible to most biochemical assays

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