DNApulldown.ppt

lrpA
amy
sipA
lrpA
amy LrpA
sipA
-25 -20
-15 -10(kDa)
Others
• Proteins fractionation methods
– Fragment DNA
F1
F2
176bp R1
F3 R2
ATG
528bp
R3
– Poly dI-dC, Salmon DNA, Calf DNA, Herring DNA
2. Incubate Beads-DNA with 2.5-5mg/ml of cell extract 100ul for 30min at 70 oC on a heater.
3. Gently shake and suspend the beads every 2-3 mins to avoid beads precipitation. After incubation, remove the supernatant ( extra cell extract) .
DNA Pull-down Protocol
Group Meeting 06-21-2005
DNA Pull-down Protocol
Dynabeads streptavidin
+
Bioinylated DNA fragment DNA containing ge百度文库e promoter sequence
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
100-
incubate with Pf cell
extract ,No recombinant
75-
LrpA added
50-
8 16
37-
❖Natural LrpA was identified
2520-
1510(kDa)-
LrpA
Natural LrpA identification
6mg/ml proteins
A 0.1MKCl vs
B 0.5M KCl
AB AB
AB AB
AB
AB
Wash 1st
Wash 2nd
Recombinant LrpA
Wash 3rd DNase digest
Final elute
Salt concentration in washing
Wash
DNase elute
KCl
A 0.4M B 0.2M A B
Identification and characterization
Bead sustain temperature
DynaBeads endurable temperature
30min heating
60oC 70oC 75oC
80oC 85oC
90oC
streptavidin
Incubation Temperature
• Different methods
– Cell extract+promoter DNA competitor DNA – Cell extract+promoter DNA + competitor DNA – Cell extract+competitor promoter DNA
DNA immobilization on DynaBeads
– Add freshly made 100mM DTT to reaction solution to make final concentration of DTT at 1mM.
• Steps
1. Wash Beads-DNA with incubation buffer 100ul/time for 3 times.
3. Twice quick wash for beads-DNA with 1x B&W buffer, 100ul/time
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
• 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
1. 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 )
2. 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.
DNA immobilization
DNA –cell extract Incubation 70oC/55oC
Wash
Elute proteins
70oC 55oC 250k150k100k-
75k-
50k37k-
25k20k-
15k10kDa-
Recombinant LrpA
Salt concentration for incubation
Thermosome single subunit
LrpA
LrpA
Different protein amount
Identification of natural LrpA from Pf cell extract
Cell extract (mg/mL) 2
150-
300 bp promoter DNA