DNA-PAINT KITMASSIVE-sdAB 2-PLEX
Expiration after 6 months

CONTENT(For research use only)

SECONDARY SINGLE DOMAIN ANTIBODIES·FluoTag®-XM-QC Anti-Mouse IgG kappa light chain (Clone: 1A23) + Docking site 1 (To be measured with·FluoTag®-XM-QC Anti-Rabbit IgG (Clone: 10E10) + Docking site 2 (To be measured with Imager 2)·Concentration: 5 µM Protein, 5 µM DNA (1 DNA strand per protein)·Volume: 100 µL·Storage: -20 °C·Storage buffer: PBS, 50 % glycerol + 0.05 % NaN3·Recommened dilution: 1:200 - 1:500 (for optimal results the dilution needs to be optimized depending on theImager 1)

target accessiblity and expression level)
IMAGERS·Imager 1 Cy3B or ATTO 655·Imager 2 Cy3B or ATTO 655·

Concentration: 1 µM in TE buffer (10 mM Tris, 1 mM EDTA, pH 8)·

Volume: 300 µL·Storage: 20 °C (1 µM imager solutions are stable for multiple freeze-and-thaw cycles)
Optional: Prepare 50 µL aliquots and store 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 plastictubes.

BUFFERS·Antibody incubation buffer, 30 mL, store at 2 - 8 °C
Note: For longer-term storage we recommend to store aliquots at -20 °C.

SAMPLE PREP.
PROTOCOL·

Washing buffer (10×), 20 mL, store at room temperature (to be diluted 1:10 in water before use)

·Imaging buffer, 50 mL, store at room temperature

1. Prepare sample using a protocol optimized for your target and primary antibody staining.
2. Wash with washing buffer (1×).
3. Dilute secondary DNA-PAINT single domain antibodies in Antibody incubation buffer.
4. Incubate for 30 - 60 min at room temperature.
5. Wash three times with washing buffer (1×).
6. Optional: Incubate fiducial markers.
7. Wash with imaging buffer before adding the final imaging solution with imager strands.
8. Before imaging: Add imager strands diluted in imaging buffer. We recommend a starting concentration of 1nM. However, the optimum imager concentration strongly depends on the target and labeling density. Thus, theimager concentration should be adjusted such that distinct single molecule blinking events can be observed.
9. After imaging, exchange buffer to washing buffer (1×) for storage at 4 °C.

IMAGINGPARAMETERS·Exposure time: 100 - 150 ms·

Laser-Intensity: ~200 W/cm2 (561 nm) and ~300 W/cm2 (640 nm). This intensity might vary due to different
illumination depths/modes (TIRF/HILO). For dense targets we recommend increasing the laser power to enhanceblinking.

·Total imaging time/target: 30 min (Depends on target density and applied imager concentration)·Temperature: The kit is optimized for image acquisition at 21 - 25 °C. At higher temperatures shorter exposure
times and higher laser powers are required.

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