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
Isolate DNA binding proteins
Non-DNA sequence specific proteins
Elute DNA sequence specific protein
Identification and characterization
3
Bead sustain temperature
DynaBeads endurable temperature
30min
heating
60oC
85oC
75oC
80oC
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
DNase elute
Wash
KCl
B
A
A 0.4M
B 0.2M
Thermosome single subunit
LrpA
LrpA
7
Different protein amount
Identification of natural LrpA from Pf cell
extract
2
8
16
Cell extract (mg/mL)
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 extractpromoter DNA competitor DNA
• Cell extractpromoter DNA competitor DNA
• Cell extractcompetitor promoter DNA

F2
F3
F1
528bp
R1
R2
176bp
R3
ATG
10
DNA immobilization on DynaBeads

• DNA length
• 300 base pairs
• Buffer - 2x BW 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
  BW 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 BW buffer, incubate for
  15mins (lt1kb DNA) under room temperature (RT).
  Keep the beads shaking gently to prevent
  precipitation in solution.
• Twice quick wash for beads-DNA with 1x BW
  buffer, 100ul/time

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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-specific DNA binding proteins

• Buffer
• Incubation buffer
• Steps
• 3 times of quick wash for beads bound DNA-protein
  complex with 100ul washing buffer _at_ 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).