Phasing assays

Imaging WT HP1-DNA liquid droplets

Materials:

WT HP1α purified according to Larson e. al. Nature, 2017

WT HP1α-KCK Cy3 purified and fluorescently labeled according to Larson et al., Nature, 2017 and Keenan et al., eLife, 2020

601 DNA or any DNA 
mPEG-Silane (Laysan Bio)

Greiner Sensoplate™ glass bottom plates 384 wells #M4437 
2% Hellmanex

0.5 M NaOH

95% EtOH

100mg/mL BSA (filtered)

Day 1:

Dialyze WT HP1α, WT HP1α-KCK into 1L assay buffer:

HP1α assay buffer: 20mM HEPEs, pH 7.5 RT, 70mM KCl, 1mM DTT Glass passivation with mPEG-silane – step 1:

1. Wash wells 2X with 100μls ddH₂O
2. Add 100μls of 2% Hellmanex and let sit for 30 mins-1 hour
3. Wash wells 3X with 100μls ddH₂O
4. Add 100μls of 0.5M NaOH and let sit for 30 minutes
5. Wash wells 3X with 100μls ddH₂O
6. Add 50μls of 20mg/mL mPEG-Silane dissolved in 95% ethanol. Cover wells with sticky foil tape and seal with end of pen Let sit overnight, and keep away from light
    

Day 2:

Take HP1α and HP1α-KCK out of dialysis and check concentrations using the Nanodrop. Glass passivation with mPEG-silane – step 2:

1. Add 50μls of 95% EtOH. Take out ~70-90μls (depends on how much ethanol evaporated overnight), attempting to keep at least 10μls at the bottom of the well. From here on out, try to avoid keeping well dry for more than a second or two.
2. Wash 2X with 90μls 95% EtOH, taking out 90μls leaving at least 10μls in the bottom of the well.
3. Wash 3X with 90μls ddH₂O, taking out 90μls leaving at least 10μls in the bottom of the well.
4. Add 100μls 100mg/mL BSA. Let sit for 30 mins,
5. Remove 90μls. Wash 3X or more with 90μls ddH2O being careful not to leave the well dry. Leftover BSA can act as crowding agent, so wash extensively!
6. Wash 3X with 90μls assay buffer 
    

Experiments:

20μl reaction 
Conditions/components:

Typically, we use HP1α concentration around 25μM final. DNA concentration is at 0.5μM final (50ng/μl)

1:500 ratio HP1α : HP1α-KCK Cy3 (μM,  e.g. 25μΜ ΗP1α:0.05μM HP1α-KCK Cy3). Adjust according to desired fluorescent signal.
 

1. Make up 20μl reaction by adding the labeled and unlabeled HP1α, DNA, and assay buffer. Let sit at RT for 30mins.
2. Mix reaction. Remove assay buffer one well at a time and reaction to the well. Be careful not to touch the bottom of the well or let it dry for too long.
3. Let droplets settle for at least 2-3 hours before imaging via BF and epifluoresence. I use 20X and 40X objective magnification