DNA Pull-down Protocol

1. DNA Pull-down Protocol Group Meeting 06-21-2005

2. DNA Pull-down Protocol + Bioinylated DNA fragment DNA containing gene promoter sequence Dynabeads streptavidin Dynabeads bound DNA fragment Add cell extract to dynabeads, incubation Magnetic separation, remove non-DNA binding proteins Non-DNA sequence specific proteins Isolate DNA binding proteins Elute DNA sequence specific protein Identification and characterization

3. Bead sustain temperature DynaBeads endurable temperature 30min heating 60oC 75oC 80oC 85oC 90oC 70oC streptavidin

4. Incubation Temperature 55oC 70oC DNA immobilization 250k- 150k- 100k- DNA –cell extract Incubation 70oC/55oC 75k- 50k- 37k- Wash 25k- 20k- Elute proteins 15k- Recombinant LrpA 10kDa-

5. Salt concentration for incubation A 0.1MKCl vs B 0.5M KCl A B A B A B A B A B A B Recombinant LrpA Wash 1st Wash 3rd DNase digest Final elute Wash 2nd

6. Salt concentration in washing Wash DNase elute KCl A B A 0.4M B 0.2M Thermosome single subunit LrpA LrpA

7. Different protein amount Identification of natural LrpA from Pf cell extract Cell extract (mg/mL) 2 8 16 150- 300 bp promoter DNA incubate with Pf cell extract ,No recombinant LrpA added 100- 75- 50- 37- • Natural LrpA was identified 25- 20- LrpA 15- 10(kDa)-

8. Natural LrpA identification 6mg/ml proteins lrpA amy sipA -25 lrpA -20 amy LrpA -15 sipA -10(kDa)

9. Others • Proteins fractionation methods • Fragment DNA • Poly dI-dC, Salmon DNA, Calf DNA, Herring DNA • Different methods • Cell extract+promoter DNA competitor DNA • Cell extract+promoter DNA + competitor DNA • Cell extract+competitor promoter DNA F2 F3 F1 528bp R1 R2 176bp R3 ATG

10. DNA immobilization on DynaBeads • DNA length • 300 base pairs • Buffer - 2x B&W buffer • 10mM Tris,1mM EDTA,2.0M NaCl,adjust the pH to ~ 7.0 by HCl • Steps • Take 50ul beads (for example) and wash with 2x B&W buffer twice, 100ul/time. (if using more beads, scale up everything else in the following steps ) • Immobilize enough DNA (about 0.39pm 300bpDNA/ul beads) on beads in 1x B&W buffer, incubate for 15mins (<1kb DNA) under room temperature (RT). Keep the beads shaking gently to prevent precipitation in solution. • Twice quick wash for beads-DNA with 1x B&W buffer, 100ul/time

11. Protein and DNA incubation • Buffer – incubation buffer • 50mM Tris, 1mM EDTA,100mM KCl, adjust pH to ~ 7.0 by HCl, 5% Glycerol, 0.1% TritonX100 • Add freshly made 100mM DTT to reaction solution to make final concentration of DTT at 1mM. • Steps • Wash Beads-DNA with incubation buffer 100ul/time for 3 times. • Incubate Beads-DNA with 2.5-5mg/ml of cell extract 100ul for 30min at 70 oC on a heater. • Gently shake and suspend the beads every 2-3 mins to avoid beads precipitation. After incubation, remove the supernatant ( extra cell extract) .

12. Remove non-DNA-binding proteins by removing supernatant Add washing buffer 3x Remove non-specific DNA binding proteins • Buffer • Incubation buffer • Steps • 3 times of quick wash for beads bound DNA-protein complex with 100ul washing buffer @ RT RT

13. Protein eluting • Buffer • DNase reaction buffer • Laemmli buffer • Methods • (Optional, if followed by protein electrophonesis) Turbo DNase digest of Beads-DNA complex for 30min at 37oC. 40ul digestion solution composed by 4 ul DNase, 4 ul 10 x digestion buffer, 32 ul nanopure water. Shake the tube frequently to avoid sediment of beads in solution. • Add 50ul 1x Laemmli buffer, and heat at 50oC for 10min to elute most proteins. Take supernatant and for gel analysis. • Heat the Beads left in the microtube at 100oC for 5 mins in 1x Laemmli buffer 50ul. ( optional, probably nothing left on beads).