DNA-PAINT protocol
Collaborating with Massive Photonics, abberior exclusively offers DNA-PAINT kits specifically designed for MINFLUX superresolution microscopy.
MASSIVE-TAG-X2 anti-GFP optimized for MINFLUX microscopy
Introduction
These kits include ready-to-use single-domain antibody (sdAB) binders for precise DNA-PAINT imaging. DNA-PAINT is a localization-based superresolution technique that utilizes dye-labeled single-stranded DNA (ssDNA) imager strands, which transiently bind to their complementary docking strands attached to the target of interest. Ideal for achieving unparalleled resolution in nanoscale imaging!
Storage
The product is shipped at room temperature and should be stored according to the instructions provided for each component in the kit upon arrival.
Hazard and Precautionary
This kit contains sodium azide with > 0.01% which may be harmful in contact with the skin, eyes, or respiratory tract. Appropriate protective clothing such as gloves should be worn. For further information, the safety data sheet is available on abberior.shop.
Immunofluorescence staining with abberior DNA-PAINT kit for MINFLUX microscopy
The procedure below has been successfully tested with our abberior DNA-PAINT dye conjugates. It has yielded consistent results in most instances but may require further optimization for particular model organisms and targets. The following protocols describe the immune DNA-PAINT staining procedure for adherent cells expressing a GFP-tagged protein grown on glass coverslips.
Reagents, provided
Single domain antibodies (sdAbs)
- TAG-X2 anti-GFP (clone: 1H1 & 1B2) + Docking site MF3
- Concentration: 2.5 µM clone 1H1, 2.5 µM clone 1B2, 5 µM DNA (1 DNA strand per protein)
- Volume: 100 µl
- Storage buffer: PBS, 50 % glycerol + 0.05 % NaN3
- Storage: -20 °C
- Recommended dilution: 1:200 – 1:500 (for optimal results the dilution needs to be optimized depending on the target accessibility and expression level)
Imager
- Imager MF3 abberior DNA-PAINT 580 or abberior DNA-PAINT 660
- Concentration: 1 µM in TE buffer (10 mM Tris, 1 mM EDTA, pH 8)
- Volume: 300 µl each
- Storage: -20 °C (1 µM imager solutions are stable for multiple freeze-and-thaw cycles). Optional: Prepare 50 µl aliquots and store them at -20 °C. Working aliquots can be stored at 4 °C for short-term or -20 °C for long term
Note: Further dilutions should be prepared fresh before use. Low imager concentrations are not stable in plastic tubes.
Buffers
- Antibody incubation buffer, 60 ml, stored at 2 – 8 °C
Note: For longer-term storage, we recommend to store aliquots at -20 °C. - Washing buffer (10×), 20 ml, store at room temperature (to be diluted 1:10 in water before use)
- Imaging buffer, 50 ml, stored at room temperature
Required reagents; not provided
- Fixative depending on cell-line and GFP construct (e.g.: 2 % – 8 % Formaldehyde in PBS (PFA), Methanol (abs.), or 4 % PFA + 0.02 % Glutaraldehyde in PBS)
- 1x Phosphate-buffered saline pH 7.4 (PBS)
- 1x PBS supplemented with 10 mM MgCl2 (Magnesium chloride)
- BBI 150 nm gold colloid solution (SKU:EM.GC150), or Nanopartz Gold Nanoparticles 150 nm (A11-150-CIT-DIH-1-10)
- Two-part silicon glue
Staining procedure for cultured cells
- Perform the fixation of the sample using a protocol optimized for your GFP-tagged protein target.
- Block cells in Antibody incubation buffer for ~30 minutes.
- Dilute sdAbs in Antibody incubation buffer.
Note: For most applications, a dilution of 1:200 of the sdAbs is sufficient. However, staining protocols may vary with cell type and application. - Incubate for 1 h at room temperature
- Wash three times with washing buffer (1×).
- Incubate the sample for 5 min with gold particle solution, with no further dilution.
- Wash three times with 1× PBS.
- Incubate in PBS supplemented with 10 mM MgCl2 for 5 minutes.
- Wash once with imaging buffer before adding the final imaging solution with imager strands.
- Before imaging: Add imager strands diluted in imaging buffer. We recommend a starting concentration of 500 pM. However, the optimum imager concentration strongly depends on the target and labeling density. Thus, the imager concentration should be adjusted such that distinct single-molecule blinking events can be observed
- Samples can be mounted on dimple slides and sealed with two-part silicon glue for easy imaging and storage. Samples should be stored at 4 °C.
Abbreviations
MINFLUX Minimal fluorescence photon fluxes
GFP Green fluorescent protein
sdAbs Single domain antibodies, nanobodies
PBS Phosphate-buffered saline
PFA Paraformaldehyde
MgCl2 Magnesium chloride
min Minute
h Hour