Crosslinking DNA-Binding Protein to DNA&

Sunday, October 19, 2003

Description
Crosslinking DNA-Binding Protein to DNA Using a Bromodeoxyuridine-Substituted Probe

Procedure
1. Combine 10 g M13 ssDNA vector and an equal molar amount of 17 bp M13 universal primer in a microcentrifuge tube. Adjust final volume to 100 l with 1x Restriction Buffer.

2. Incubate the reaction at 37C for 5 min. Allow the primer and vector to hybridize overnight (at least 12 hrs.) at room temperature.

3. To the hybridized sample, add:

50 l [32P]dCTP (3000 Ci/mmol)

3.5 l 50x dNTP/BrdU solution

1.75 l 0.1 M DTT

7.5 l 10x Restriction Buffer

7 l ddH2O

25 U Klenow fragment

Incubate the reaction at 16C for 90 min.

4. Heat inactivate the Klenow enzyme for 10 min at 70?C.

5. Add appropriate Restriction enzyme(s) to digest the DNA to generate DNA fragments (see Hint #1).

6. Precipitate the labeled DNA: Add 0.1 volumes of 3 M Ammonium Acetate and 2 volumes of Ethanol. Mix by inversion a few times and incubate the tube on dry ice (-70C) until solid (approx. 15 min. or at -20C overnight). Pellet DNA by centrifugation in a microcentrifuge at maximum speed for 10 min. Carefully remove the supernatant and discard appropriately.

7. Resuspend the DNA pellet in 100 l of TE.

8. Prepare a 1% agarose gel in TBE or TAE, load the labeled DNA, and fractionate the DNA by electrophoresis in the agarose gel (see Protocol on Agarose Gel Electrophoresis). Isolate fragments of expected size and purify the labeled DNA from the agarose (see Protocol on Gel Purification of DNA).

9. Calculate the efficiency of labeling and specific activity of the labeled DNA.

10. Incubate the probe with extract containing DNA binding protein of interest with the following conditions:

Approximately 1 x 10E5 cpm of labeled probe to extract and add the appropriate amount of ssDNA to maximize specific binding (see Hint #2). Incubate the reaction at room temperature for the appropriate amount of time.

12. Crosslink the labeled DNA to the bound protein by exposing the reaction to UV light: Place tube with cap open in a holder on ice within a container, and place close (~10 cm or less) to the source of UV light. Irradiate for the appropriate amount of time (see Hint #3).

13. After crosslinking, digest the uncrosslinked DNA with the addition of:

1 l 0.5 M CaCl2

4 g DNase I

1 U micrococcal nuclease

Incubate the reaction at 37C for 30 min.

14. Add an equal volume of 2 X SDS-PAGE Sample Loading Buffer. Cap tubes tightly and boil for 5 min. Allow tubes to cool and load on an SDS-Polyacrylamide gel. Fractionate proteins by SDS-PAGE and analyze protein-DNA crosslinks with autoradiography (see Protocol on SDS-PAGE) (see Hint #4).

Recipes
3000 Ci/mmol -[32P]-dCTP (Caution See Hint #5)

10X Restriction Endonuclease Buffer 10 mM DTT
500 mM NaCl
100 mM Tris-Cl, pH 7.5
1 mg/ml BSA
100 mM MgCl2
Add DTT fresh before use and store at -20C

17-bp M13 universal primer

Single-stranded M13 vector with desired binding site

DNase I

0.5 M CaCl2

Salmon sperm DNA (ssDNA)

Extract containing DNA-binding protein

TE Buffer 10 mM Tris-Cl, pH 7.5
1 mM EDTA

100% Ethanol

3M Ammonium acetate

25 U Klenow fragment of E. coli DNA polymerase I

0.1 M DTT in distilled, deionized water (ddH2O). Store at -20?C.

50X dNTP/BrdU solution 250 um dCTP
2.5 mM dGTP
2.5 mM 5-bromo-2'-deoxyuridine triphosphate (BrdU)
Store at -20C.
2.5 mM dATP

Protein molecular weight standards

Tris-glycine Electrophoresis Buffer (SDS/sample buffer) 10x pH ~8.2
0.38 M Glycine
0.5 M Tris base
2 mM EDTA

2x SDS/Sample Buffer 2% (w/v) 2-Mercaptoethanol or 3.1% (w/v) DTT
125 mM Tris
20% (v/v) Glycerol
0.001% (w/v) Bromophenol Blue
4% (w/v) SDS

Micrococcal nuclease

Supplies

Tips
1. Choice of Restriction enzyme(s) will depend upon the number of sites present in the DNA vector sequence. Ideal size range is between 20 to 600 bp.

2. Optimal conditions for binding of the protein of interest to DNA must be empirically determined. Try trial experiments, varying the concentration of ssDNA (maximum of 20 g per reaction), amount of extract, and time of incubation before crosslinking.

3. Optimal crosslinking conditions are dependent on the protein of interest and factors described in Hint #2. Try varying the amount of time and distance from the UV light to maximize specific crosslinking of the protein of interest with the labeled DNA. An alternative to crosslinking in microcentrifuge tubes (e.g. because the UV light source will not allow for the size of the ice-filled container) is to place reactions on a sheet of parafilm in concave indentations in the parafilm molded into the wells of microcentrifuge tube holder racks. In this case care must be taken to minimize exposure of spillage of the radioactive material.

4. Make sure to either run the gel so that the dye runs off the bottom, or when disassembling the gel, cut off the portion at the bottom which will contain uncrosslinked, labeled DNA fragments. This will be very radioactive and should be handled and disposed of carefully. The gel should be dried before exposing to X-ray film.

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

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