Exonuclease III protection assay (updated 3/27/96): This assay is very sensitive for detecting DNA binding proteins because it does not require complete occupancy of the DNA by the protein. The DNA must be kinased on the 5' end since exonuclease III degrades 3' to 5'. The exonuclease III will displace some DNA binding proteins. For example, the assay does not detect binding of the GAGA factor. The first application of this assay to Drosophila proteins can be found in Wu, 1985, Nature 317:84-87. For results that are more pertinent to work in our lab, see Gilmour et al., 1988, MCB 8:3204-3214. In this updated procedure, the Na phosphate has been taken out of the original 2X ExoIII binding buffer because it will form an insoluble precipitate with Magnesium.

1. For a 12.5 ul binding reaction, first combine the components listed below (make an appropriate cocktail when possible to increase the uniformity of the samples). Store the tubes on ice while the reactions are being assembled. Don't forget a no protein control because exonuclease III will inherently make stops at certain sequences.
• 6.25 ul of 2X Exo III binding buffer {A recipe is provided at the end} 2X exo III binding buffer contains: 10 mM MgCl₂, 30 mM Tris pH 7.6, 10 % glycerol, 40 ng/ul HaeIII-cut E.coli DNA, 40 ng/ul random Hexamers, 400 ng/ul yeast tRNA. Note that the 4 mM Na Phosphate pH 6.8 has been eliminated from the original recipe - it is added at independently. The random Hexamers are the same as those used for random priming of DNA. They provide a large excess of ends which presumably limit phosphatase activity. Store the buffer at -20°C and be sure that everything has dissolved before using.
• 10,000 to 20,000 cpm of kinased DNA
• 0.1 ul of 100 mM DTT
• Enough 1M KCl so that the final concentration will be 80 mM after addition of protein (the protein usually contributes some KCl).
• 1.25 ul of 20 mM Na Phosphate pH 6.8 (store this stock at -20°C so nothing grows in it)
• 1 or 2 ul of protein
• Enough H₂O so that the final binding reaction after addition of Na Phosphate and protein totals 12.5 ul
2. Float tubes in a 25°C water bath. The water can be set up in any insulated box that doesn't leak. (Carefully note temperatures.  The Drosophila proteins are allowed to bind at 25°C but the exonuclease digestion is performed at 30°C.)
3. Add protein to tubes at 30 second intervals. TFIID and HSF can be easily detected when 1ul of crude nuclear extract is combined with kinased hsp26 DNA. TFIID is also easily detected on hsp70 but HSF can be difficult. Don't forget a "no protein control" where you simply add the buffer that the protein is in. Be sure to use a good pipettor like the Oxford with tips that form good seals so that accurate volumes of protein are added to each tube. Thoroughly mix each by triterating with the pipette. Avoid generating air bubbles.
4. Incubate for 30' at 25°C (Shorter times like 20 minutes can be used but be sure to be consistent for all of the samples)
5. During the binding reaction, dilute just enough exonuclease III to 20 units/ul in ice cold 50 mM NaCl, 20 mM Tris-Cl pH8, 5 mM MgCl₂. 2 ul of this diluted exonuclease III is needed for each binding reaction. Store on ice until needed.
6. Set up a second water bath at 30°C.
7. At the end of each binding reaction, add 2 ul of the diluted exonuclease III and mix by triteration. Avoid generating air bubbles.
8. Place the sample at 30°C for 10'; work quickly because the samples are staggered by 30 seconds.
9. After 10 minutes at 30°C, stop the exonuclease III digestion by adding 25 ul of 10 mM EDTA (pH 7.5-8), 0.5 % Sarkosyl {or see protocol provided by Phil Walter and described towards the end}
10. Add 5 ug of proteinase K and digest at 37°C for at least 15'.
11. Add water to a final volume of 100 ul.
12. Add 100 ul of phenol/chloroform/isoamyl alcohol mix (49.5/49.5/1, sometimes called leder phenol) and shake vigorously for 1'. Centrifuge for 5' at 20°C. Remove the underlying organic layer with a pipette and discard it in the phenol waste bottle in the hood.
13. Ethanol precipitate the DNA. Add 10 ul of 3M Na Acetate pH~5.5 and 250 ul of ethanol. Mix thoroughly by inverting the tube and then incubate on ice for 15'. Centrifuge at 4°C for at least 20'. A small pellet should be visible due to the nonspecific nucleic acid that is present in the binding reaciton. Remove as much as possible of the supernatant with a drawn-out glass pipette. Air dry sample for 10'.
14. Dissolve the precipitate in sequencing gel loading buffer. Slosh the buffer around extensively by vigorously snapping the sides of the tube. Boil for 3', quench on ice and load onto the sequencing gel. Be sure to have a set of markers and don't forget the "no protein control".

Recipe for 1 ml of 2X ExoIII buffer:

• 2.5 ul of 4M MgCl₂
• 30 ul of 1M Tris-Cl pH 7.6
• 200 ul of 50% Glycerol
• 10 ul of 4 ug/ul HaeIII-cut E. coli DNA
• 8 ul of 5 ug/ul random hexamers
• 20 ul of 20 ug/ul yeast tRNA
• 729.5 ul of H₂O

Alternative isolation of DNA after digestion provide by Phil Walter.

1. Stop ExoIII reaction by adjusting the volume to 50 ul and having the following final concentrations of components:
• 20 mM Tris-Cl pH 8.0
• 50 mM EDTA
• 2% SDS
• 200 ug/ml proteinase K
• 0.25 mg/ml yeast tRNA
2. Incubate for at least 1 hour at 50°C
3. Add 25 ul of 7.5M Ammonium Acetate, mix well
4. Add 150 ul of Ethanol, mix well
5. Ice for 1 hour
6. Centrifuge for 30'
7. Wash with 75% ethanol
8. Dry and dissolve in sequencing gel loading buffer.