Product Catalog

Use the DIG Gel Shift Kit to prepare nonradioactive, 3´-end-labeled oligonucleotide probes, so they can be used to detect DNA-protein complexes in a "gel mobility shift assay." 

• 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 

The study of DNA-protein interactions has been greatly facilitated in recent years by the rapid and simple “gel retardation” or “gel mobility shift” assay. Because free DNA and DNA-protein complexes migrate differently during gel electrophoresis, they can be separated and detected on native (non-denaturing) polyacrylamide or agarose gels.

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

The DIG Gel Shift Kit uses probes that are end-labeled with digoxigenin-11-ddUTP to detect sequence-specific DNA-binding proteins.

The labeled DNA fragment that contains the sequence of interest is incubated with the cell extract or a purified factor. Nonspecific DNA [poly d(A-T), poly d(l-C), provided in the kit] is added to the extract to prevent the formation of large nonspecific protein-DlG-labeled DNA complexes (which would remain at the top of a gel and cause smearing during gel electrophoresis).

The mixture is transferred to a native polyacrylamide gel and electrophoretically separated. Following the separation, the oligonucleotide-protein complexes are transferred onto positively-charged nylon membranes by electroblotting, capillary transfer, or contact blotting.

Digoxigenin-labeled complexes on the membrane are detected in an immunoassay. An alkaline phosphatase-conjugated antibody [anti-digoxigenin-AP (Fab fragments)] binds the digoxigenin-labeled oligonucleotides.The immobilized alkaline phosphatase dephosphorylates the chemiluminescent substrate CSPD. During dephosphorylation, CSPD emits light, which can be recorded on an X-ray film. Chemiluminescent signals can usually be detected after the membrane is exposed to the film for 15 - 40 min. (See "Typical Experiment" below.)

Major stages in procedure:

1. DIG 3´-end labeling of double-stranded oligonucleotides with recombinant Terminal Transferase
2. Gel shift reaction, in which DIG 3´-end-labeled oligonucleotides are incubated with cell extract or a purified factor
3. Polyacrylamide gel electrophoresis, using the PhastSystem (Pharmacia)
4. Transfer of oligonucleotides onto nylon membranes (blotting)
5. Crosslinking 
6. Chemiluminescent detection 

Figure 1: Detection of digoxigenin-labeled oligonucleotides.

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.

The electrophoresis assay works best with shorter (30 - 100 bp) DNA fragments, since these are less likely to have sequences that are outside the specific binding site, but can interact nonspecifically with target proteins.