Spot-Label for Immunofluorescence

Catalog No. ABIN6810488

Product Details

Target Name (Antigen):

Spot-Tag

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 Details

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

Handling

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 Details

Target Name (Antigen):

Spot-Tag

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