Sunday, October 19, 2003

Description
The protocol is designed to detect protein-DNA complexes. The DNA is radiolabeled then mixed with the protein. The protein binding can be detected because the protein-DNA complexes have a slower mobility in a polyacrylamide gel than the free DNA

Procedure
A. Making the Band Shift Probe

1. Digest the plasmids containing regions of DNA to be used as the probes with the appropriate Restriction Enzymes .

2. End label the DNA fragments with -[32P]-ATP .

3. Gel-purify the labeled DNA fragment.

B. Binding Reaction

1. Combine the following:
(putative) DNA-binding Protein
1,000 to 5,000 cpm of radiolabeled DNA
4 l of 5X Reaction Binding Buffer
ddH2O to a final volume of 20 l

2. Set up a series of reaction tubes with increasing amounts of protein (up to 10 pmole) to titrate the amount of protein needed to shift the free probe.

3. Incubate for 60 min at room temperature.

C. Electrophoresis of Shifted DNA

1. Prepare a 4% Polyacrylamide Gel :
6.7 ml of 30% Acrylamide Solution
1.25 ml of 20X TBE
500 l of 10% Igepal CA-630
41.6 ml of ddH2O

Just before pouring the gel add:
250 l of 10% Ammonium Persulfate
60 l of TEMED

2. Use 1.5 mm spacers between the gel plates.

3. After the gel has polymerized, allow the gel to cool at 4C in a cold room.

4. Add 1 l of 6X Loading Dye to 5 l of Protein:DNA mixture.

5. Load the Protein:DNA mixture onto the gel.

6. Electrophorese the Protein:DNA mixture at 4C and 150 V in 0.5X TBE until the Bromophenol Blue dye travels approximately one-half to three-quarters the distance to the bottom of the gel.

7. Disassemble the gel and transfer it to solid support for drying (e.g., Whatman 3MM paper).

8. Dry the gel.

9. Autoradiograph the gel to analyze shifted protein:DNA complexes.


Recipes
Reaction Binding Solution (5X) 5 mM DTT
500 ng Polydeoxyinosinic-Deoxycytidylic Acid (dIdC) (used as competitor DNA)
125 mM HEPES-NaOH, pH 7.6
0.25% (v/v) Igepal CA-630
750 mM NaCl
All concentrations given are the final reaction concentrations.
5 mM PMSF
1 mM EDTA
50% (v/v) Glycerol


Phenylmethylsulfonyl Fluoride (PMSF) Stock Prepare in DMSO (CAUTION! See Hint #1)
250 mM PMSF


Loading Dye (6X) 0.3% (w/v) Bromophenol Blue
60% (v/v) Glycerol
0.3% (w/v) Xylene Cyanol FF


10% Ammonium Persulfate Suspend in ddH2O
10% (w/v) Ammonium Persulfate


10% Igepal CA-630 Suspend in ddH2O
10% (w/v) Igepal CA-630


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


30% Acrylamide Solution 30% (v/v) Acrylamide (60:1 Acrylamide:Bis-Acrylamide; CAUTION! See Hint #1)
Suspend in ddH2O



Supplies


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

2. For P-element band shifts, use probes that correspond to the ends of the P-element.

3. For other proteins, you can adjust the incubation time, salt concentration, amount of competitor DNA, and different detergents.