Protocols for DIG-gel shift assays

Dec. 30, 2008

Procedures

Annealing and labeling of oligonucleotides

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Mix solutions of complementary oligonucleotides in TEN-buffer in a molar ratio of 1:1.
Incubate for 10 min at 95C.
Cool slowly to 15-25C.
Dilute with sterile TEN-buffer to 3-4 pmol/l.
Add 3.85 pmol double strand oligonucleotide and sterile double distilled water to a final volume of 10 l to a reaction vial.
For the control reaction, add 1 l control oligonucleotide and 9 l sterile double distilled water to a reaction vial.
Add the following on ice:
5x labeling buffer 4 l
CoCl2-solution   4 l
DIG-ddUTP solution 1 l
Terminal transferase 1 l
Mix and centrifuge briefly.
Incubate for 15 min at 37C, then place on ice.
Stop the reaction by adding 2 l 0.2 M EDTA (pH 8.0).
Add 3 l double distilled water to a final volume of 25 l, to obtain a final concentration of 4 ng/l or 0.155 pmol/l of the labeled oligonucleotide.

Determination of labeling efficiency

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Apply a 1 l spot of tubes 1-5 from your labeled oligonucleotide and the labeled control to the nylon membrane.
Fix the nucleic acid to the membrane by cross linking with UV-light or baking for 30 min at 120.
Transfer the membrane into a plastic container with 20 ml of washing buffer. Incubate under shaking for 2 min at 15 to 25C.
Incubate in 10 ml of blocking solution for 30 min.
Incubate in 10 ml of antibody solution for 30 min.
Wash with 10 ml of washing buffer, 2 x 15 min.
Equilibrate 2-5 min in 10 ml of detection buffer.
Place membrane with DNA side facing up on a development folder (or hybridization bag) and apply 0.1 ml of CSPD® working solution.
Immediately cover the membrane with the second sheet of the folder to spread the substrate evenly and without air bubbles over the membrane.
Incubate for 5 min at 15 to 25C. Squeeze out excess liquid and seal the edges of the development folder.
Note: drying of the membrane during exposure will result in dark background.
Incubate the damp membrane for 10 min at 37C to enhance the CSPD chemiluminescent reaction.
Expose to X-ray film or imaging device.
Note: luminescence continues for at least 48 hours. The signal increases in the first few hours after initiation of the detection reaction until it reaches a plateau where signal intensity remains almost constant during the next 24-48 hours.
Multiple exposures can be taken to achieve the desired signal strength.

Gel shift reaction

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Mix on ice:
Binding buffer
DIG-labeled oligonucleotide
Protein
Double distilled water
Mix carefully and incubate for 15-30 min at 15-25.
Place tube on ice.
Add to each sample 5 l of loading buffer with bromophenol blue.
Apply samples immediately to a pre-electrophoresed polyacrylamide gel.

Polyacrylamide gel electrophoresis

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One day before
Prepare a native polyacrylamide gel of 4% or 6% acrylamide in 0.5 x TBE buffer.
Note: prepare the gel the day before use to make sure that the gel is completely polymerized.
The gel must be pre-run.
Load the samples to the gel.
Note: before loading samples, clean sample wells to remove APS, urea and residual polyacrylamide to ensure sample application without diffusion.
Run a 10 cm x 10 cm x 0.1 cm PAGE at 80 V.
Note: for other gel sizes use 8 V/cm.
Run dye 2/3 of the way to the bottom of the plates.

Blotting and crosslinking

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After electrophoresis: remove one glass plate carefully from the gel.
Equilibrate a sheet of nylon membrane trimmed to the size of the gel for 5 min in transfer buffer (0.5x TBE buffer).
Place equilibrated nylon membrane carefully onto the gel.
Note: avoid air bubbles between gel and filter.
Place 4 layers of gel-sized Whatman 3MM papers, presoaked in transfer buffer on the filter.
Roll with a glass rod or a pipette over the 4 layers Whatman 3 MM paper to remove air bubbles.
Remove pad of Whatman 3MM paper/nylon membrane/gel from the other glass plate.
Add 4 layers of pre-soaked Whatman 3MM papers on the other side of the gel.
Place resulting sandwich between the electrodes of the electroblotting device.
Perform transfer for a 10 cm x 10 cm x 0.1 cm PAGE for 30 min at 400 mA (NOVEX System: 60 min; 30 V; 300 mA).

Crosslinking of oligonucleotides

Bake at 120C: bake the membrane for 15-30 min
Or:
Place the membrane on a Whatman 3 MM paper presoaked with 2x SSC, cross-link at 120 mJ in e.g. a stratalinker or with a transilluminator for e.g. 3 min which has to be tested empirically.

Chemiluminescent detection

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Rinse membrane briefly (1-5) min in washing buffer.
Incubate for 30 min in 100 ml of blocking solution.
Incubate for 30 min in 20 ml of antibody solution.
Wash 2 x 15 min in 100 ml of washing buffer.
Equilibrate 2-5 min in 20 ml of detection buffer.
Place membrane with DNA side facing up on a development folder (or hybridization bag) and apply 1 ml CSPD working solution.
Immediately cover the membrane with the second sheet of the folder to spread the substrate evenly and without air bubbles over the membrane.
Incubate for 5 min at 15 to 25C.
Squeeze out excess liquid and seal the edges of the development folder.
Note: Drying of the membrane during exposure will result in dark background.
Incubate the damp membrane for 10 min at 37C to enhance the luminescent reaction.
Expose to X-ray film for 15-25 min or imaging device at 15C25C.
Note: luminescence continues for at least 48 hours. The signal increases in the first few hours after initiation of the detection reaction until it reaches a plateau where signal intensity remains almost constant during the next 24-48 hours.
Multiple exposures can be taken to achieve the desired signal strength.

Materials

Washing buffer: 0.1 M maleic acid, 0.15 M NaCl, pH 7.5 (20C); 0.3% (v/v) Tween-20, 15 to 25C, stable.
Maleic acid buffer: 0.1 M maleic acid, 0.15 M NaCl, adjust with NaOH (solid) to pH 7.5 (20C), 15 to 25C, stable.
Detection buffer: 0.1 M Tris-HCl, 0.1 M NaCl, pH 9.5 (20C), 15 to 25C, stable.
TEN-buffer: 10 mM Tris, 1 mM EDTA, 0.1 M NaCl, pH 8.0, 15 to 25C, stable.
Blocking stock solution: Dissolve blocking reagent 10% (w/v) in maleic acid buffer under constantly stirring on a heating block (65C) or heat in a microwave oven, autoclave. The solution remains opaque. Stable for 4 weeks at 2 to 8C if kept sterile.
1x Blocking solution: Prepare a 1x working solution by diluting the 10x blocking solution 1:10 in maleic acid buffer. Always prepare fresh.
Antibody solution: Centrifuge Anti-Digoxigenin-AP for 5 min at 10,000 rpm in the original vial prior to each use, and pipet the necessary amount carefully from the surface. Dilute Anti-Digoxigenin-AP 1:10 000 (75 mU/ml) in blocking solution. And 12 h at 2 to 8C binding to the DIG-labeled probe.
CSPD working solution: Dilute 0.1 mg/ml stock solution 1:100 in detection buffer, and 2 to 8C protected from light chemiluminescent detection.