Advanced surface passivation for high-sensitivity studies of biomolecular condensates

Significance The understanding of biomolecular condensates has significantly benefited from biochemical reconstitution with microscopy detection. Here, we present a unique surface passivation method utilizing self-assembly of Pluronic F127 on hydrophobic surfaces. This approach not only effectively minimizes nonspecific binding without altering the physical properties of the condensates but also offers passivation across a variety of condensate systems. It demonstrates high resistance to different treatments and enables condensate immobilization through controlled anchor points. This allows for highly sensitive analytical techniques, including single-molecule imaging. The simplicity and high performance of this method, coupled with time and cost efficiencies, could facilitate robustness and throughput of experiments, and could broaden the accessibility of biochemical phase separation studies to a wider scientific community.


Supporting Information Text
Detailed protocol to passivate glass surface by Pluronic F127 self-assembly for in vitro biomolecular condensate studies Reagents For all subsequent preparations outlined in this protocol, 'MilliQ H2O' refers to water with a resistivity of at least 18.2 MΩ cm and a total organic carbon (TOC) content of no more than 5 ppm.

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Allow the plate to air dry in the hood for about 10 min.Note: Oil-like residues may appear but will be washed away in subsequent steps.

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Rinse the plate twice with isopropanol.

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Fully dry the plate in the hood.

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Cover the entire dried plate with PCR foil.The plate can be kept for at least a few months.
The following 4 and 5 sections describe two methods for assembling Pluronic F127.The first method, passivation without anchor points, is appropriate for routine phase separation assays, such as visualizing fusion or constructing phase diagrams.The second method, involving passivation with anchor points, is designed for immobilizing droplets during prolonged imaging processes like 3D imaging, FRAP, and single molecule tracking.
4. Assembling Pluronic F127 without Anchor Point  Remove foil from required wells using a scalpel.

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Fill wells with 100 µL 0.5%(w/v) Pluronic F127 in buffer that will be used in phase separation assay and incubate for > 15min.

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After incubation, sequentially wash each well with buffer used in phase separation assay for at least 5 times (sequentially wash means first remove 90 µL buffer, then add 90 µL fresh buffer, then remove 90 µL buffer and repeat for at least 5 times.DO NOT dry the glass at any time or the assembled F127 will be destroyed.One can also wash the well by suck up the buffer using a vacuum system while adding 500 µL wash buffer by pipette). Discard the excess solution but leave ~5 µL buffer in the well.DO NOT dry the well. Note: Although drying the wells should generally be avoided, it has been observed that some types of condensates can maintain their integrity on dried glass.

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Wells are now ready for the phase separation assay.
5. Assembling Pluronic F127 with anchor point  Remove foil from required wells using a scalpel.

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Fill wells with 100 µL 0.5%(w/v) Pluronic F127 in buffer that will be used in phase separation assay.Incubate for > 15min.

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Sequentially wash each well with the buffer at least 5 times.Avoid drying the glass.(See section 4 for details)  Incubate wells with 20 µL 15 nM NeutrAvidin in buffer that will be used in phase separation assay for 5 minutes at room temperature.Wash wells with 200µl buffer.DO NOT dry the glass at any time.

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Discard the excess solution but leave ~5 µL buffer in the well.DO NOT dry the well.

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Wells are now ready for the phase separation assay.Note: Add a small amount of biotin-labeled phase separation protein when preparing your sample.About 1% biotin-labeled protein is usually sufficient to immobilize condensates with low wettability.For sticky condensates, Biotin-NeutrAvidin-Biotin interaction may not be necessary.Incubating with BSA before F127 assembly will be enough to create sticky regions for immobilizing condensates.

Fig. S1 .
Fig. S1.Comparative analysis of time and cost for PF127 and mPEG/BSA passivation.A, Workflow and time cost comparison between PF127 passivation and mPEG/BSA passivation.Image created with BioRender.com.B, Comparison of financial cost of PF127 passivation versus mPEG/BSA passivation.Note that the calculated costs may vary depending on the distributor and region.

Fig. S2 .
Fig. S2.Characterization of homogeneity of PF127 passivation.Homogeneity comparison of PF127 and mPEG/BSA passivation in millimeter-scale fields of view.

Fig. S3 .
Fig. S3.Characterization of auto-fluorescence background of PF127 passivation.A, Auto-fluorescence comparison of PF127-passivated glass with bare glass under 405 nm, 488 nm, 552 nm and 638 nm laser excitation.Emission light from 430 to 770 nm is monitored with a 20 nm bandwidth.The autofluorescence levels are compared with the fluorescence signals from

Fig. S4 .
Fig. S4.Characterization of phase separation behavior of chromatin condensates under PF127 passivation.A, Phase separation behavior of chromatin condensates with 25 bp and 30 bp linker lengths at 1 μM nucleosome concentration on PF127-passivated glass.B, Partial and whole droplets FRAP of 25 bp linker chromatin condensates on PF127 passivated glass (mean ± SD, n=11 condensates for partial FRAP, n=5 condensates for whole droplets FRAP).
Place the plate on a clean table.Rinse the beaker, fill with 1L tap water, and submerge the plate, well side up.Rotate for 10 s.  Repeat the rinsing step 10 times with tap water. Repeat the rinsing step another 10 times, this time using MilliQ H2O.If using a plastic syringe, ensure it's compatible with Sigmacote. Dispense about 100µL Sigmacote into the first well, then immediately drain.Continue for each well, ensuring complete coverage.The reaction is almost instantaneous so you can process this step very fast as long as Sigmacote covers glass surface.
2. Etching glass with hydroxide Tap inverted plate on a clean paper towel to remove excess water.Filleachwell with 100 µL 1 M KOH.Let it sit for 1 hour at room temperature.