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Spot-Label for Immunofluorescence Accessory Reagents

Recombinant bivalent alpaca single-domain antibody, ATTO 594 conjugated, for the analysis of Spot-Tag fusion proteins.
Catalog No. ABIN5680071
$103.13
Plus shipping costs $45.00
10 μL ABIN5680071
10 μL ABIN5680071
local_shipping Shipping to: United States
Delivery in 5 to 6 Business Days
  • Target Name (Antigen)
    Antibody Type
    Recombinant Antibody
    Reactivity
    Tag
    Host
    Camelid (Camelidae)
    Conjugate
    Atto 594
    Application
    Immunofluorescence (IF)
    Purpose
    Recombinant bivalent alpaca single-domain antibody, ATTO 594 conjugated, for the analysis of Spot-Tag fusion proteins.
    Brand
    Spot-Tag®
    Specificity
    Spot-Label binds to the Spot-Tag sequence PDRVRAVSHWSS at the N-terminus or the C-terminus. Tested in cell culture.
    Characteristics
    Spot-Label for epifluorescence, widefield, confocal, and super-resolution microscopy of Spot-tagged proteins. Spot-Label is an anti-Spot-Nanobody (anti-Spot-VHH, Spot-binding protein) conjugated to fluorescent dyes. For immunofluorescence (IF) microscopy, a bivalent format of the Spot-Label is used: two anti-Spot-Tag VHHs/Nanobodies connected by a linker and labelled with fluorophores.
    Purification
    Purified
    Components
    Spot-Label ATTO594 for Immunofluorescence (IF), bivalent
    Excitation Emission
    Excitation range 580 - 615 nm (λabs= 601 nm), Emission range 620 - 660 nm (λfl= 627 nm)
  • Application Notes
    For immunofluorescence (IF) microscopy, a bivalent format of the Spot-Label is used: two anti-Spot-Tag VHHs connected by a linker and labelled with fluorophores.
    IF: 1/2,000
    Optimal working concentration is application-dependent and should be determined by testing a range of dilutions from 1,000 to 5,000.
    Comment

    Small peptide tags are useful for the labelling and detection of proteins using immunostaining, immunoblotting or immunoprecipitation techniques. Spot-Tag is a short 12 amino acid affinity tag PDRVRAVSHWSS, which can be cloned either N- or C-terminally to a protein of interest. This tag can be efficiently immunostained with the novel Spot-Label affinity reagent. The Spot-Label consists of a small recombinant bivalent alpaca single-domain antibody fragment covalently coupled to a fluorescent dye. Due to its small size, immunostaining of the Spot-Tag with the Spot-Label minimizes the ""linkage error"" for super-resolution microscopy applications (e.g. STED and dSTORM). In addition, the Spot-Label has a superior tissue penetration rate, better access to the Spot epitope, and higher labelling density.

    Reagent Preparation

    Fixation buffer: 3.7 % formaldehyde in PBS
    Permeabilization buffer: PBS, 0.5 % Triton X-100
    Wash buffer: PBS
    Blocking buffer: 4 % BSA (w/v), PBS

    Assay Procedure
    1. Fixation: Fix cells seeded on coverslips in 3.7 % formaldehyde in PBS for 10 min at room temperature.
      Note: Always prepare a fresh formaldehyde dilution.
      2. Wash samples three times with PBS (Phosphate Buffered Saline). Do not store fixed cells.
      3. Permeabilization: Add PBS containing 0.5 % Triton X-100 to samples and incubate for 5 min at room temperature.
      Note: Alternatively, use ice-cold 100 % methanol for permeabilization.
      4. Wash samples twice with PBS.
      5. Blocking: Add 4 % BSA in PBS to samples and incubate for 20 min at room temperature.
      Note: If necessary, use additional blocking reagents (e.g. 10 % normal serum in PBS or Image-iTTM FX Signal Enhancer from ThermoFischer Scientific) and extend the blocking time up to 60 min.
      6. Spot-Label incubation: Dilute Spot-Label 1:2000 in blocking buffer and incubate for overnight at +4 °C.
      Note: For multiplexing protocols, you can combine Spot-Label with another primary or secondary antibody.
      7. Wash samples three times for 5-10 min in PBS.
      8. If required, counterstain with DNA fluorescent dyes, e.g. DAPI in PBS. Proceed with imaging directly or mount samples, if necessary.
      9. Mounting: Rinse sample briefly in water to prevent salt crystal formation. Mount in ProLongTM Diamond Antifade Mountant from ThermoFischer Scientific or other mounting media with anti-fading agents.
    Restrictions
    For Research Use only
  • Format
    Liquid
    Concentration
    1 g/L
    Buffer
    1 x PBS, 0.09 % sodium azide
    Preservative
    Sodium azide
    Precaution of Use
    This product contains Sodium azide: a POISONOUS AND HAZARDOUS SUBSTANCE which should be handled by trained staff only.
    Handling Advice
    Do not freeze. Protect from light.
    Storage
    4 °C
    Storage Comment
    refrigerate 4°C
    Expiry Date
    6 months
  • Target Name (Antigen)
    Target Type
    Tag
    Background
    The Spot-Tag has an affinity-optimized 12 amino acid sequence to be strongly bind to the Spot-Nanobody. The inert tag can be bound to the N-terminal and to the C-terminal side of the protein of interest by PCR using modified primers or by using any of the Spot vectors offered. The expression of Spot-tagged proteins has been successfully tested in bacteria, yeast, mammalian cell lines, and insect cells so far.

    Features:
    - Small size
    - PDRVRAVSHWSS
    - 12 amino acid sequence
    - 1.4 kDa
    - pI: 10.4
    - Hydrophilic
    - Inert
    - Linear
    - Extinction coefficient: 5,500 L•mol-1•cm-1

    Benefits:
    - Easy to clone
    - Works N-terminally and C-terminally
    - Good expression
    - Tested in bacteria, yeast, mammalian cell lines, and insect cells
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