• Convenient, since the technique does not require special equipment or technology
• Reproducible, since DIG-labeled probes are stable indefinitely
• Safe, because the assay is nonradioactive
• Reliable, because the kit provides DIG-labeled control oligonucleotides to establish that the assay is working 
• Function-tested with the controls provided in the kit (See "Quality" below.) 
• Sensitive, since the kit can detect as little as 20 fmol of the control oligonucleotide (after it is labeled according to the kit protocol)

Brief description
The kit contains reagents for nonradioactve 3´-end-labeling of oligonucleotides, so they can be used in a "gel mobility shift" assay. It also contains controls, electrophoresis reagents, and an enzyme-labeled antibody to facilitate the detection of DNA-protein complexes.
Note: The combination of recombinant Terminal Transferase and DIG-11-ddUTP makes the labeling reaction very flexible. It can be used to label the 3-ends of any oligonucleotide (whether it has a 5- overhanging end, a 3-overhanging end or blunt ends). Both single- and double-stranded DNA can be labeled.

Sample
Amount: 3.85 pmol or 100 ng 
Type: Oligonucleotides with 5´-overhanging ends, 3´-overhanging ends, or blunt ends;
single- or double-stranded DNA fragments (30 - 200 bp)
Note: Ideally, fragments to be labeled should be between 30 and 100 bp. (See "Notes" below for more details.)

Time required
Oligonucleotide annealing and labeling: 10 min
Formation of oligonucleotide-protein complexes: 25 min
Electrophoresis: 1 h to overnight, depending on electrophoresis system
Blotting: 1 - 2 h
Immunological detection: 2 h
Exposure to X-ray film or imager: 15 - 40 min 

1. Labeling Buffer (5x conc.), 80 µl
2. CoCl₂ Solution (25 mM), 80 µl
3. DIG-ddUTP Solution (1 mM Digoxigenin-11-ddUTP), 20 µl
4. Recombinant Terminal Transferase (400 U/µl), 20 µl
5. Binding Buffer (5x conc.), 800 µl
6. Control Oligonucletide (ds 39mer, unlabeled, 0.1 µg/µl, 3.85 pmol/µl), 40 µl
7. DIG-labeled Control Oligonucleotide (ds 39mer, 0.4 ng/µl, 15.54 fmol/µl), 140 µl
8. Control Factor Oct2A (25 - 75 ng/µl), 40 µl 
9. Poly [d(I-C)] (0.1 µg/µl), 200 µl
10. Poly [d(A-T)] (0.1 µg/µl), 200 µl
11. Poly L-lysine (0.1 µg/µl in double-distilled water), 200 µl
12. Loading Buffer without bromophenol blue, 1 ml
13. Loading Buffer with bromophenol blue, 1 ml
14. Anti-Digoxigenin-AP (750 U/ml), 40 µl
15. CSPD (10 mg/ml), 440 µl
16. Blocking Reagent, 50 g

Function test: Half (50%) of the control oligonucleotide is shifted by the control factor under the conditions described in the protocol. The assay can detect 20 fmol of control oligonucleotide after it is labeled according to the kit protocol.