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DNA Pull-down Protocol. Group Meeting 06-21-2005. DNA Pull-down Protocol. +. Bioinylated DNA fragment DNA containing gene promoter sequence . Dynabeads streptavidin. Dynabeads bound DNA fragment. Add cell extract to dynabeads, incubation.

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dna pull down protocol

DNA Pull-down Protocol

Group Meeting

06-21-2005

slide2

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

bead sustain temperature
Bead sustain temperature

DynaBeads endurable temperature

30min

heating

60oC

75oC

80oC

85oC

90oC

70oC

streptavidin

incubation temperature
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-

salt concentration for incubation
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

salt concentration in washing
Salt concentration in washing

Wash

DNase elute

KCl

A

B

A 0.4M

B 0.2M

Thermosome single subunit

LrpA

LrpA

different protein amount
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)-

natural lrpa identification
Natural LrpA identification

6mg/ml proteins

lrpA

amy

sipA

-25

lrpA

-20

amy

LrpA

-15

sipA

-10(kDa)

others
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

dna immobilization on dynabeads
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
protein and dna incubation
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) .
remove non specific dna binding proteins

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

protein eluting
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).