Spot-Label® for epifluorescence, widefield, confocal, super-resolution microscopy and Western blotting of Spot-tagged proteins. Spot-Label is an anti-Spot-Nanobody (anti-Spot-VHH, Spot-binding protein) conjugated to fluorescent dyes. A bivalent format of the Spot-Label is used: two anti-Spot-Tag® VHHs/Nanobodies connected by a linker and labelled with fluorophores.
Spot-Label binds to the Spot-Tag sequence PDRVRAVSHWSS at the N-terminus or the C-terminus.
Tested in cell culture.
- Immunofluorescence (IF)
- Western blot (WB)
Optimal working concentration is application-dependent and should be determined by testing a range of dilutions.
Purified recombinant protein in PBS Size: 10μL or 50μL
0.5 – 1mg/ml (conjugates)
0.09% sodium azide
Shipped at ambient temperature. Upon receipt store at 4°C. Do not freeze. Protect from light.
Widefield images of HeLa cells expressing Actin-Chromobody-EGFP-Spot-Tag immunostained with Spot-Label ATTO594, bivalent (1:5,000). Left: GFP channel, middle: channel for ATTO594, right: overlay, also with DAPI. Scale bar, 10 µm.
Confocal images of HeLa cells expressing Tom70-EGFP-Spot-Tag immunostained with Spot-Label ATTO594 bivalent (1:1,000). Left: EGFP fluorescence, middle: ATTO594 signal, right: merge. Scale bar, 10 µm. The images were acquired with Leica TCS SP8 microscope, 100X oil objective, at the Core Facility Bioimaging at the Biomedical Center, LMU Munich, and deconvolved with Huygens Professional (SVI). Left: GFP fluorescence, middle: ATTO594 signal, right: merge. Scale bar, 10 µm.
Super resolution microscopy
STED: IF of Spot-tagged Actin-Chromobody with Spot-Label Atto594 bivalent (1:1,000). Gated STED images were acquired with a Leica TCS SP8 STED 3X microscope with pulsed White Light Laser excitation at 590 nm and pulsed depletion with a 775 nm laser. Objective: 100x Oil STED White, NA: 1.4. Pixel size: 21 x 21 nm; z-Step size of z-Stacks: 0.16 µm. Images were deconvolved with Huygens Professional (SVI). STED images were recorded at the Core Facility Bioimaging at the Biomedical Center, LMU Munich.
Western blot with Spot-Label ATTO488 (left) and Western blot with Spot-Label ATTO594 (right)
For Spot-Tag cloning information and available Spot vectors click here.