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206 Urh et al.
3. Incubate for 10 min at room temperature.
4. Quench crosslinking by the addition of glycine, pH 7, to a final concentration of
125 mM directly to cells.
5. Incubate for 5 min at room temperature.
6. Aspirate off media and wash cells twice with 2 ml of ice-cold 1× PBS.
7. Add 1.5 ml of ice-cold PBS to cells and scrape cells into a 1.5-ml microcentrifuge
tube.
8. Place cells immediately on ice.
9. Centrifuge and pellet cells at 2000 × g for 5 min at 4°C.
10. Remove PBS and resuspend cells in 650 μl of lysis buffer.
11. Vortex and incubate on ice for 15 min.
12. Dounce cells or lyse them by passing them through 25–27-guage needle several
times using 1-ml syringe.
13. Sonicate on ice to obtain DNA fragments between 500–1000 bp (see recommendations
below for a Misonix 3000 sonicator).
14. Clear lysates by centrifugation at 14000 × g for 10 min at 4°C.
15. Add lysate (supernatant) directly to prepared HaloLink resin and incubate with
rotation for 2hatroom temperature or 4–18 h at 4°C.
16. Spin lysates with HaloLink resin at 800 × g for 3 min. Discard supernatant.
17. Wash resin twice with 1 ml of lysis buffer. Discard supernatant each time.
18. Wash resin twice with 1 ml with high salt lysis buffer. On the last wash, incubate
resin with buffer for 5 min at room temperature with rotation. Discard supernatant
each time.
19. Wash resin three times with 1 ml nuclease-free water. On the last wash, incubate
resin with water for 5 min at room temperature with rotation. Discard supernatant
each time.
20. Add 100 μl of reversal buffer to resin and place tubes at 65°C for 4–18 h to
reverse crosslinks.
21. Centrifuge resin at 800 × g for 3 min after reversal and save the supernatant
containing released target DNA.
22. Purify DNA for PCR amplification using a PCR clean-up kit according to
manufacturer’s recommendations.
Misonix 3000 Sonication Recommendation (Microtip 418): Set the output
to 2.5. For 1×10 6 cells in a volume of 500–700 μl, on ice, perform 6 × 10-s
pulses with 10 s of rest in between each pulse.
3.5. Enzyme Immobilization and Analysis of Enzymatic Activity
on the Surface
Immobilization of enzymes and study of their enzymatic activities is very
important. Covalent attachment of proteins to the HaloLink resin allows
assaying of enzymatic activities over a long period of time in different buffer
conditions without protein dissociation from the resin. Affinity purification