Determination of the Molecular Weight of a DNA Binding Prote

Sunday, October 19, 2003

Description
This protocol describes a procedure for determining the molecular weight of a DNA-binding protein. First, DNA containing a binding site (or multiple sites) for the protein of interest is labeled with [32P]. The labeled DNA and extract containing the protein of interest are then mixed together to allow binding. Following binding, the DNA-protein complexes are crosslinked with UV irradiation. The crosslinked complexes are digested to remove any DNA that is not protected by the protein binding and the mixture is then run on an SDS polyacrylamide gel. Autoradiography is used to determine the molecular weight of the specifically bound protein.

Procedure
A. Primer-Template Annealing (see Hint #1)

1. Combine the following components:

1 l of 1 g/l single stranded DNA from an M13 clone

1 l of 10X M13 Annealing Buffer

5 l of 0.5 pmole/ l M13 Universal Primer

3 l of ddH2O

2. Incubate for 10 min at 65C.

3. Continue to incubate at room temperature for 30 min.

B. Labeled Strand Synthesis

1. Add to the above incubation solution:

2 l of 10X Cold dNTPs (see Hint #2)

5 l of 10 mCi/ml -[32P]-dCTP (800 Ci/mmol or 3,000 Ci/mmol, CAUTION! see Hint #3 and see Hint #4)

1 l of 100 mM DTT

2 l (4 Units) of Klenow Fragment (Boehringer-Mannheim)

2. Incubate for 1 hr at 37C.

3. Add 1 l of 0.5 mM dCTP to chase.

4. Incubate at 37C for 30 min.

C. Restriction Digest

1. Add to above reaction:

4 l of 10X High Salt Restriction Buffer

15 Units of EcoRI (see Hint #5)

15 Units of PstI (see Hint #5)

Bring the final volume to 40 l using ddH2O.

2. Incubate for 1 hr at 37C.

D. Preparative Gel Electrophoresis and Band Excision

1. Add 7 l of 6X DNA Loading Dye to the reaction.

2. Load sample onto an 1.5 mm thick 8% Acrylamide Native DNA Prep Minigel prepared in 0.5X TBE.

3. Electrophorese at 300 V until the Bromophenol Blue dye migrates approximately two-thirds down the gel (about 1 hr).

4. Wrap the gel in Saran Wrap and add radioactively-labeled ink dots as alignment guides.

5. Autoradiograph the gel by exposing the gel to film for approximately 1 min (see Protocol on Autoradiography).

6. Properly align the film to the gel, determine the position of the radiolabeled bands, and remove the radiolabeled band (this should be the lower radiolabeled band).

E. OPTION #1: Isolation of DNA by Electroelution from Acrylamide Gel (see Hint #6)

1. Use forceps to place the excised band of radiolabeled DNA into a dialysis bag filled with 0.5X TBE.

2. Electroelute 3 to 4 times for 30 min at 150 V (see Hint #7; see Protocol on DNA purification by Electroelution).

3. To the aqueous phase add an equal volume of Phenol (CAUTION! see Hint #3), mix well by inversion, centrifuge for 3 min in a microcentrifuge to separate the phases, and save the upper, aqueous phase.

4. To the aqueous phase add an equal volume of SEVAG, mix well by inversion, centrifuge for 3 min in a microcentrifuge to separate the phases, and save the upper, aqueous phase.

5. Place the DNA sample into 0.5" x 2" SW50.1 polyallomer tubes and add 5 M NaCl to a final concentration of 0.1 M.

6. Add 3 volumes of 100% Ethanol, cover the tube with Parafilm and mix carefully.

7. Chill at -80C for approximately 30 min (or overnight).

8. Centrifuge at 35,000 rpm using SW 50.1 rotor (115,000 X g) for 20 min at 4C.

9. Remove the supernatant, vacuum dry the pellet in either a vacuum centrifuge or a lyophilizer and resuspend in 80 l TE Buffer, using two 40 l aliquots.

10. Determine the radioactivity of a 1:10 dilution of the resuspend DNA. There should be greater than 5,000 cpm; it is best to have above 10,000 cpm and a specific activity of 107 cpm/g DNA.

11. Store the radiolabeled DNA at -20C.

F. OPTION #2: Isolation of DNA by the "Crush and Soak" Method

1. Push the gel slice through a 3 ml plastic syringe 5 times to mash the gel into small fragments.

2. Place 3 Whatman GF/C glass fiber filters, cut to the correct size using a cork borer, in the bottom of a 5 ml syringe .

3. Place the gel fragments into the syringe.

4. Place Parafilm over the bottom of the 5 ml syringe and place it in 17 x 100 mm polypropylene tube.

5. Add 0.5 ml of Crush and Soak Buffer and cover the tube with Parafilm.

6. Incubate at 37C for 2 to 3 hr on a platform shaker rotating at approximately 200 rpm.

7. Centrifuge for 1 to 2 min at approximately 1,500 X g in a clinical centrifuge.

8. Remove the eluate to a 1.5 ml microcentrifuge tube and add 10 g of Glycogen and 2.5 volumes of 100% Ethanol.

9. Chill the samples at -80C for approximately 30 min.

10. Centrifuge for 5 min at maximum speed in a microcentrifuge to pellet the DNA.

11. Discard the supernatant and resuspend the DNA pellet in 200 l of TE Buffer.

12. Add 25 l of 3 M Sodium Acetate and 750 l of 100% Ethanol. Repeat Steps #9 and #10.

13. Discard the supernatant and resuspend the DNA pellet in 80 l TE Buffer, using two 40 l aliquots.

14. Determine the radioactivity of a 1:10 dilution of the resuspended DNA. There should be greater than 5,000 cpm; it is best to have above 10,000 cpm and a specific activity of 107 cpm/g DNA.

15. Store the DNA at -20C.

G. Crosslinking Reaction

1. The Protein-DNA binding reaction should contain 1 l of -[32P]-BrdUTP-substituted probe in 10 l. Use conditions suitable for the protein of interest, e.g. the conditions used for DNAse I footprinting (see Hint #8).

2. After the binding reaction, open the microcentrifuge tube and expose the solution to 302 nm UV light for 15 min at room temperature or spot the solution onto a piece of Parafilm set on a cold block and expose to 302 nm UV light for 10 min (see Hint #9 and #10).

3. Digest away the excess nucleic acid by adding 1 l of Nuclease Salt Solution and 1 l of Nuclease Enzyme (see Hint #11).

4. Incubate at 37C for 10 to 30 min.

5. Add 12 l of 2X SDS Gel Sample Buffer.

6. Boil the sample for 3 min and then load on an SDS polyacrylamide gel (see Protocol on Polyacrylamide Gel Electrophoresis).

8. Electrophorese the gel.

9. Dry the SDS gel and subject to autoradiography.

Recipes
10% Ammonium Persulfate Stable for approximately 3 weeks at 4C
10% (w/v) Ammonium Persulfate

30% Acrylamide 30% (w/v) of 30:0.8 Acrylamide:Bisacrylamide (CAUTION! see Hint #3)

High Salt Restriction Buffer (10X) 500 mM NaCl
0.25% (v/v) Triton-X 100
100 mM MgCl2
1 M Tris-Cl, pH 7.5

0.5 mM dCTP

100 mM DTT

Cold dNTPs (10X) 0.5 mM dATP
0.5 mM 5-Bromo-2'-Deoxyuridine 5'-Triphosphate (BrdUTP)
0.5 mM dGTP

M13 Anealing Buffer (10X) 0.1 M Tris-Cl, pH 7.5
66 mM MgCl2
0.6 M NaCl

TE Buffer 10 mM Tris, pH 8.0
1 mM EDTA

3 M Sodium Acetate

70% Ethanol 70% (v/v) Ethanol

5 M NaCl

Nuclease Enzyme Solution 0.1 to 2 Units Micrococcal Nuclease (RNase and protease-free)
0.5 to 3 g DNase I

Nuclease Salt Solution 0.1 M CaCl2
0.1 M MgCl2

Crush and Soak Buffer 0.1% (w/v) SDS
1 mM EDTA
10 mM Magnesium Acetate
0.5 M Ammonium Acetate

SEVAG 24:1 Chloroform:Isoamyl Alcohol (CAUTION! see Hint #3)

TBE (20X) 40 mM EDTA
1.78 M Tris
pH 8.0
1.78 M Boric Acid

DNA Loading Dye (6X) 0.25% (w/v) Xylene Cyanol
0.25% (w/v) Bromophenol Blue
40% (w/v) Sucrose

8% Native DNA Prep Gel 10.7 ml of 30% Acrylamide
6 l of TEMED
29 ml of ddH2O
1 ml of 20X TBE
0.2 ml of 10% Ammonium Persulfate

SDS Gel Sample Buffer (2X) 200 mM DTT
Add DTT just before use
20% (v/v) Glycerol
100 mM Tris-Cl, pH 6.8
0.2% (w/v) Bromophenol Blue
4% (w/v) SDS

Supplies

Tips
1. The protocol uses single stranded DNA containing 4 to 6 binding sites prepared from recombinant virions. To construct the recombinant, multimerize oligonucleotides containing a protein binding site and ligatable BamHI ends with T4 DNA Ligase and clone into the BamHI site of M13mp8 and/or replicative form (RF) DNA.

2. BrdU is misincorporated by the polymerase in place of Thymidine. DNA with substituted BrdU more easily forms a photoadduct because the Bromide atom is more susceptible to free radical formation when exposed to UV radiation.

3. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.

4. The nucleotide containing the label can be varied. If the protein binding site is AT rich, then better transfer of label might be observed by labeling with -[32P]-dATP. Remember to adjust the cold nucleotide mix accordingly.

5. EcoRI and PstI are used to cut out the fragment because some restriction enzymes will not cut after BrdU is substituted into the DNA.

6. This option is messy as it involves electrophoresing radioactive samples through agarose gels. .

7. The electroelution should recover approximately 90% of the DNA from the band.

8. The conditions may need to be determined empirically, starting with the conditions used for a DNase I footprinting reaction. DO NOT USE POLYVINYL ALCOHOL IN THE REACTION.

9. If using a hand-held UV lamp held in close proximity to the sample, that is, approximately 3 mm from the protein-DNA solution, make sure the Parafilm is laying on a metal block on ice so that the solution doesn't evaporate during the crosslinking procedure.

10. Exposure of the DNA-protein complexes to UV light causes the formation of covalent bonds between the DNA and nearby amino acids of the bound protein. Amino acids that can participate in this reaction are Arginine, Cysteine, Histidine, Lysine, Methionine, Serine, Phenylalanine, Trytophan, and Tyrosine.

11. The amount of nuclease may need to be adjusted for your protein. In general, use the least amount needed to give a sharp band on a SDS polyacrylamide gel to preserve the signal.
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Determination of the Molecular Weight of a DNA Binding Prote