Mapping of protein binding sites on DNA by DNAase I footprinting assay
Mapping of protein binding sites on DNA by DNAase I footprinting assay
This protocol describes a mapping method for identifying protein binding sites on radiolabeled DNA fragments. The protocol uses DNAase I to cut the DNA, whereas in the alternative protocol the DNA is broken with hydroxyl radicals.For advice on optimizing the footprinting reaction, see the Troubleshooting section at the end of the protocol and the section on optimizing the footprinting reaction with DNAase I. The protocol is based on the results of a molecular cloning experiment performed by J. Sambrook D. Sambrook. This experiment was derived from the next volume of the Laboratory Guide to Molecular Cloning (Third Edition) by [American] J. Sambrook D.W. Russell.
Operation method
Mapping of protein binding sites on DNA by DNAase I footprinting assay
Materials and Instruments
Fresh tissue, cultured cells, protein components from cells or tissues Move makings For more product details, please visit Aladdin Scientific website.
Cell homogenization buffer Cell resuspension buffer Cell lysis buffer Ethanol Ficoll 400 Formamide stain MgCl2 CaCl2 solution NaCl NonidetP-40 Phenol: Chloroform Phosphate buffer Polyvinyl alcohol terminator Tissue homogenizer Tissue resuspension buffer Tissue blue dye DNA enzyme I Denaturing polyacrylamide sequencing gel poly(dI-dC) Sequencing gel length Standard 32P end-labeled DNA
Beckman SW28 Turn Tables or Equivalents Sorvall Hl000B Turn Tables or Equivalents Boiling Water Bath Dounce Screeder with Type B Mortar and Pestle Crystalline Plastic or Ultra-Bright Bucket Centrifuge Tubes Rubber Rods
Buffers and solutions
See Appendix 1 for the composition of storage solutions, buffers and reagents Storage solutions should be diluted to the appropriate concentration.
Cell homogenization buffer
10 mmol/LHEPES-KOH (pH 7.9)
1.5 mml/LMgCl2
10 mmol/LKCl
0.5 mmol/LDTT
0.5 mmol/L phenylmethylsulfonyl fluoride
Cell homogenization buffer with 0.05% (V/V) NonidetP-40
Cell Resuspension Buffer
40 mmol/LHEPES-KOH (pH 7.9)
0.4 mol/LKCl
1 mmol/LDTT
10% (V/V) glycerol
0.1 mmol/L phenylmethylsulfonyl fluoride
0.1%(m/V) proteolytic peptide
Buffer stored at 0°C
Cell Drench Buffer
40 mml/L Tris-Cl (pH 7.4)
1 mmol/LEDTA
0.15 mol/LNaCl
buffer stored at 0°C.
Ethanol
Ficoll 400 (20% m/V)
Ficoll is dissolved in sterile water, dispensed into 100ul and frozen at -20°C.
Formamide Staining Solution
10 ml Formamide Gel
10 mg Xylene Cyanine FF
10 mg Bromophenolan
Store at room temperature.
MgCl2/CaCl2 solution
10 mmol/LMgCl2
5 mmoI/LCaCl2
solution was filtered for sterilization and stored at room temperature.
NaCl (5 mol/L)
NonidetP-40 (0.05%V/V)
Phenol: chloroform
Ca2+, Mg2+ free phosphate buffer solution (PBS)
Polyvinyl alcohol (10% m/V)
Polyvinyl alcohol is dissolved in sterile water, dispensed into 100ul and frozen at -20°C.
Termination Solution
20 mmol/LEDTA (pH 8.0)
1% (m/V) SDS
0.2 mol/LNaCl
125ug/ml yeast tRNA
Tissue homogenate
10 mmol/LHEPES-KOH (pH 7.6)
25 mmol/LKCl
0.15 mmol/L arginine
0.5 mmol/L peryleneamine
1 mmol/LEDTA(pH8.0)
2 mol/L sucrose
10% (V/V) glycerol
The buffer needs to be pre-cooled in an ice bath before use. Protease inhibitors such as 0.5 mmol/L phenylmethylsulfonyl fluoride (PMSF), 1ug/ml leupeptin, 1ug/ml pepstatin, etc., are added before step 1 as needed.
Tissue Resuspension Buffer
5 mmol/LHEPES-KOH(pH7.9)
1.5 mmol/LMgCl2
0.5 mmol/LDTT
0.5 mmol/L phenylmethylsulfonyl fluoride
26%(V/V) Glycine max
Store at 0°C.
Taipan Blue Dye (0.4% m/V)
Dissolve a quantity of the dye in Ca2+ and Mg2+ free phosphate buffer solution (PBS). Store at room temperature.
Enzymes and buffers
DNAase I (lmg/ml)
Enzyme is dissolved in 10 mmol/L Tris-Cl (pH 8.0), dispensed into small portions, and frozen at -20°C. Dilute the solution 1:100 in 10 mmol/L Tris-Cl (pH 8.0) in an ice bath just prior to step 3.
Gel
6% or 8% denaturing polyacrylamide sequencing gels (see Scheme 8 in Chapter 12)
Nucleic acids and oligonucleotides
poly(dI-dC)(lmg/ml)
A quantity of poly(dI-dC) is dissolved in sterile water, dispensed in lOOul portions, and frozen at -20°C. Nucleic acid polymers are added to minimize nonspecific binding of proteins to radiolabeled DNA fragments, and the optimal concentration of poly(dI-dC) in the binding reaction (typically 0-100ug/ml) needs to be determined empirically. Other nucleic acids that can be used to minimize non-specific binding include sheared genomic DNA (e.g., from E. coli, salmon spermatozoa, or calf thymus), tRNA, sheared or restriction digested plasmid DNA, poly(dA-dT), and poly(dG-dC).
Sequencing gel length standards
Typically consists of the (A+G) reaction in a Maxam-Gilbert sequencing assay of a target DNA fragment. For chemical sequencing, see Scheme 7 in Chapter 12; alternatively, the sequencing reaction can be performed using the double deoxy termination method (Schemes 3-6 in Chapter 12), where the 5' end of the DNA used is the same as the radioactive end of the DNA fragment cut by DNAase I. The DNA is then sequenced to the 5' end of the target DNA fragments.
Radioactive compounds
32P end-labeled DNA [length 200-500bp, specific activity ≥2. 5X107cpm/ug (≥5000cpm/fmol)].
End-labeling of DNA fragments can be accomplished by phosphorylation (Scheme 13-16 in Chapter 9, or Scheme 2 in Chapter 10); complementation of 3'-indented ends with DNA polymerase (Schemes 10 and 11 in Chapter 9, and Scheme 7 in Chapter 10); or PCR reactions with end-labeling primers (Scheme 1 in Chapter 8). The latter method is faster, does not depend on restriction sites, and allows the DNA binding site to be located at more than one position in relation to the end-labeling. Regardless of the method of radiolabeling, the DNA fragments need to be purified by agarose or polyacrylamide gel electrophoresis prior to use in the DNA footprinting reaction.
The length of the DNA fragments used in the reaction should be 200-500 bp. The binding site of interest should be at least 30 bp from the end of the radiolabel. if longer DNA fragments are used, the resolution of the sequencing gel will be reduced. Binding sites too close to the end of the dam may not be recognized by DNA-binding proteins or by DNAase I. The binding site should be at least 30 bp from the end of the radiolabel.
Centrifuges and Turntables
Beckman SW28 turntable or equivalent, precooled to 4°C
Sorvall Hl000B Turning head or equivalent, pre-cooled to 4°C
Specialty Equipment
Boiling water bath
Dounce homogenizer with type B mortar and pestle
Crystal Plastic or Ultra-Bright Bucket Centrifuge Tubes
Rubber Stick
Auxiliary reagents
Steps 7 to 10 of this program require the reagents listed in Chapter 12, Programs 8, 11 and 12.
Cells and Tissues
Fresh tissue, cultured cells, protein components from cells or tissues 
Methods
1. Prepare a nuclear extract by one of the following 3 methods. In addition, the fractions obtained by purifying cellular proteins can be used directly in step 2.
Preparation of nuclear extracts from tissues
a. Dissect 10-15 g of tissue and cut into pieces. Add ice-cold Tissue Homogenization Buffer to bring the volume of minced tissue to 30 ml. Homogenize with a Dounce homogenizer with a tight-fitting mortar and pestle until microscopic examination determines that >80% to 90% of the cells have been broken.
b. Lysis is detected by adding 10ul of cell suspension to the same volume of 0.4% Taipan Blue Stain and observing the solution under a microscope fitted with a 20x objective. Lysed cells absorb the stain and are colored blue, while intact cells are unstained and remain clear. Continue tissue homogenization until >80%~90% of the cells are broken.
C. Dilute homogenate with ice-cold Tissue Homogenization Buffer to 85 ml. Place 10 ml of ice-cold Tissue Homogenization Buffer in a crystalline plastic or ultra-bright boom-type centrifuge tube and spread 27 ml of diluted homogenate on top. Centrifuge at 103,900 g (24,000 r/min with a Beckman SW28 turntable) at 4°C for 40 min.
d. Remove the supernatant and invert the tube for 1 to 2 min to allow the solution to run dry. The tubes are then placed on ice.
(Optional) Cut off the top 2/3 of the tube with a razor blade and place the remaining 1/3 with the nuclei on ice.
e. Resuspend the nuclei precipitate with 2 ml of ice-cold Tissue Resuspension Buffer. Accurately measure the volume of the resuspended nuclei solution and add ice-cold 5 mol/L NaCl to a final concentration of 300 mmol/L. Gently mix the suspension and ice bath for 30 min.
The suspension is gently mixed and then ice-bathed for 30 min. f. Recover the nuclei by centrifugation at 103,900 g (24,000 r/min with a Beckman SW28 turntable) at 4°C for 20 min. g. The supernatant is carefully transferred to fresh water. Carefully transfer the supernatant into fresh tubes. Dispense the supernatant into small portions of 100 to 200 ul. Leave one portion for protein concentration determination. The others were snap-frozen in liquid nitrogen and stored in liquid nitrogen.
g. Determine the protein concentration of the supernatant by the Bradford method.
Preparation of nuclear extracts from cultured cells
a. Harvest 0. 5X108 to 1X108 cells from culture flasks, culture blood or culture wells. Collect cells by centrifugation at 250 g (1100 r/min with Sorvall Hl000B turntable) at room temperature. Cells were washed several times with Ca2+ -free phosphate buffer.
b. Resuspend the cell sediment with 5 times the volume of ice-cold cell homogenization buffer containing 0.05% (V/V) Nonidet P-40. Ice bath for 10 min, then collect by centrifugation as before.
c. Resuspend the cell precipitate with 3 times the volume of ice-cold Cell Homogenization Buffer containing 0.05% (V/V) Nonidet P-40, and homogenize the cells by 20 strokes with a Dounce homogenizer with a tight-fitting mortar and pestle. The homogenizer is buried in ice during the homogenization process when the cells swell and lyse and release intact nuclei.
d. Collect nuclei by centrifugation at 250 g (1100 r/min with Sorvall H1000B turntable) at 4°C, remove the supernatant, and resuspend the nuclei precipitate with 1 ml of Cell Resuspension Buffer. The volume of resuspended nuclei solution was measured accurately and ice-cold 5 ml/L NaCl was added to a final concentration of 300 mmol/L. The suspension was gently mixed and then ice-bathed for 30 min.
e. Recover the nuclei by centrifugation at 103,900 g (24,000 r/min with a Beckman SW28 turntable) at 4°C for 20 min. Carefully transfer the supernatant to a pre-cooled fresh tube. Dispense the supernatant into small portions of 100 to 200 ul. Leave one portion for protein concentration determination. The others were snap-frozen in liquid nitrogen and stored in liquid nitrogen. The protein concentration of the supernatant was determined by the Bradford method.
Preparation of nuclear extracts from small amounts of cultured cells
This method is applicable to cells transfected with plasmids that express cDNAs encoding transcription factors.
a. Cells are washed several times with cell drench buffer. Add 1 ml of cell drench buffer to each petri dish and scrape the cells into the buffer with a rubber mallet.
b. Transfer the cell suspension to a 1.5 ml centrifuge tube and centrifuge the cells at maximum speed for 2 min at room temperature.
c. For cells originally cultured in each 150 mm diameter Petri dish, resuspend the cells with 300ul of Cell Resuspension Buffer, followed by 3 freeze-thaws.
d. Remove cell debris by centrifugation at 4°C for 5 min at maximum speed. The supernatant (i.e. cell lysate) is stored at -70°C in small portions.
2. Add to a 1.5 ml centrifuge tube:
Nuclear extract or protein fraction 1-23ul
32P end-labeled DNA 1~10fmol
1 mg/ml poly(dI-dC) 1ul
H2O add to 25ul
Optional addition:
20% Ficoll 400 12ul
or
10% Polyvinyl Alcohol 10ul
Centrifuge at 4°C for about 5s to bring all the reaction solution to the bottom of the tube. Ice bath for 10-30 min. 
3. Add 50ul of MgCl2/CaCl2 solution and mix gently at room temperature. Allow to stand at room temperature for 1 min. Add 1-8 dilutions of DNAzyme I solution, mix gently and allow to stand at room temperature for 1 min.
4. Terminate the reaction with 75ul of termination solution. Shake quickly and extract the reaction solution with the same volume of phenol/chloroform.
5. Transfer the liquid phase to a new centrifuge tube and precipitate the nucleic acids with 2.5 times the volume of ethanol. The ethanol solution is allowed to stand at -70°C for 15 min, then centrifuged at maximum speed for 10 min at 4°C to collect the precipitate. The precipitate is washed with 1 ml of 70% ethanol, centrifuged again, and dried in air to remove residual ethanol.
6. Add 5~10ul of formaldehyde dye and shake vigorously to dissolve the DNA precipitate. Boil for 3-5 min to denature the DNA solution.
7. Prepare 6% or 8% denatured polyacrylamide sequencing gel and pre-electrophoresis for at least 30 min before adding samples.
8. Add the DNA samples in the following order:
Sequence Ladder
Control DNA digested with DNAase I in the absence of nuclear extracts.
Target DNA digested with DNAase I in the presence of nuclear extracts
Target DNA digested with DNAase I in the presence of nuclear extract but without DNAase I.
9. Run the gel at a constant power sufficient to maintain a temperature of 45 to 50°C.
The run time for optimal resolution of the sequence of interest should be determined empirically. The progress of electrophoresis is observed from the mobility of the dye in the formaldehyde gel sample buffer.
10. At the end of electrophoresis, pry off the glass plate and transfer the gel to a piece of heavy hybridization paper. Vacuum dry the gel for about 1 h, and then expose it to X-ray film, or if a sensitizing screen is not used, expose it at -20°C for 12-16 h. In addition, the dried gel can be analyzed by phosphorimage analysis, which takes about 1-3 h. The gel is then exposed to X-rays at -20°C for about 1 h. The gel is then exposed to X-rays for about 1 h. 
