Binding Assay

Stepwise, the assay is divided into (1) the pulldown procedure; (2) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE); and (3) Western blot analysis. Materials for each procedure are described separately.

2.3.1. Pulldown Procedure

1. Streptavidin immobilized on 4% beaded agarose activated by cyanogen bromide (Sigma).

2. Single-stranded 5'-biotinylated oligonucleotides (Integrated DNA Technologies, Coralville, IA) (see Note 1).

3. PBSI buffer.

4. Rocking platform labquake (Barnstead International, Dubuque, IA).

5. Microcentrifuge.

1. 2X Laemmli sample buffer: 950 pL of Laemmli sample buffer mixed with 50 pL of 2-mercapthoethanol (Bio-Rad).

2. 4 to 15% Gradient polyacrylamide gel in Tris-HCl (Bio-Rad).

3. 10X Running buffer containing 250 mM Tris, 1.92 M glycine, 1% SDS (all from Sigma). 1X Running buffer is prepared by diluting 100 mL of 10X buffer with 900 mL deionized water.

4. 10X Transfer buffer containing 250 mM Tris-HCl, 1.92 M glycine (both from Sigma). Stored at RT. 1X Transfer buffer is prepared by mixing 100 mL of 10X transfer buffer with 200 mL of methanol and 700 mL of deionized water.

5. Methanol (AAPER Alcohol and Chemicals Company, Shelbyville, KY).

6. Prestained broad-range SDS-PAGE standards (Bio-Rad).

7. Mini-Trans Blot Electrophoretic Transfer Cell (Bio-Rad).

8. Minigel holder cassette (Bio-Rad).

10. Trans-Blot Transfer Medium Supported Nitrocellulose Membrane, 0.2 pm (Bio-Rad).

11. Filter paper (Bio-Rad).

2.3.3. Western Blot

1. Rabbit affinity-purified polyclonal antibodies specific for transactivators and coac-tivators (various sources). Nonimmune IgG control (various sources) (see Note 2).

2. IgG-horseradish peroxidase-conjugated polyclonal antibody (see Note 3).

3. Blotting grade blocker nonfat dry milk (Bio-Rad).

5. Rocking platform Labquake (Barnstead International).

6. Materials for detection of proteins by chemiluminescence:

a. Kodak X-Omatic Cassette (Eastman Kodak, Rochester, NY).

b. Kodak BioMax MS film (Eastman Kodak).

c. X-ray film processor Konica SRX-201-A (Source One Healthcare Technologies, Mentor, OH).

d. SuperSignal® West Pico Chemiluminescent Substrate kit (Pierce) (see Note 4).

e. Rocking platform Labquake (Barnstead International).

f. Plastic Sheet (Reports Cover, C-Line Products, Mount Prospect, IL) (see Note 5).

3. Methods

3.1. Nuclear Extract Isolation

1. All procedures are done on ice or in a controlled temperature room at 4°C.

2. Remove medium from cultured cells and wash them once with cold PBS. Add PBSI buffer (1 mL/10-cm dish) and harvest cells with a rubber scraper. Cells are collected into a 50-mL conical tube, which is centrifuged at 550g for 5 min (see Note 6).

3. Remove supernatant. Transfer the pellet to a 1.5-mL microcentrifuge tube, and centrifuge at 1500g for 30 s.

4. To buffers A, B, and D, add the following inhibitors: 0.5 mMPMSF, 1 mMNa3VO4, 0.5 mM DTT, 1 yg/mL leupeptin, 25 mM P-glycerophosphate, 10 mM NaF.

5. Remove supernatant and resuspend the pellet in 2 package cell volume of buffer A with inhibitors. Keep on ice for 10 min. Vortex briefly, and centrifuge at 2600g for 30 s.

6. Remove supernatant and resuspend the pellet in 2/3 package cell volume of buffer B with inhibitors.

7. Sonicate the mixture for 5 s. Centrifuge at 10,400g for 5 min (see Note 7).

8. Dilute the supernatant isovolumetrically with buffer D with inhibitors. Aliquot and store at -80°C (see Note 8).

3.2. Protein Assay

1. Prepare standard albumin concentrations at 50, 100, 250, 500, 750, 1000, 1500, and 2000 yg/mL.

2. Pipet 10 yL of standards and samples each in duplicate onto a 96-well plate.

3. Mix 50 parts of buffer A with 1 part of buffer B.

4. Add 200 yL of mixed buffer A and B to standards and samples.

5. Incubate plate at 37°C for 30 min.

6. Read the protein content at 562 nm on a microplate spectrophotometer.

3.3. Binding Assay

3.3.1. Pulldown Procedure

1. Dissolve single-stranded biotinylated oligonucleotides in deionized sterile water. The final concentration is 1 yg per 1 to 2 yL.

2. Add an equal quantity of sense and antisense biotinylated oligonucleotide solution to a microfuge tube, mix, and place it in a 100°C water bath for 1 h. Remove the tube and allow it to cool down at RT (see Note 9).

3. Gently mix the streptavidin-agarose bead suspension with a vortex and place the suspension in a 1-mL (tuberculin) syringe. Pull the syringe piston forward and backward gently two to three times to ensure an even suspension (see Note 10).

4. Add 2 drops of the streptavidin-agarose bead suspension to a mixture of 400 pg of nuclear extract proteins and 4 pg of double-strand biotinylated oligonucleotides in 500 pL of PBSI buffer (see Note 11).

5. Place the mixture on a rocking platform at RT and rock the mixture at a gentle speed for 2 h (see Note 12).

6. Centrifuge the mixture at 550 g for 1 min (see Note 13).

7. Discard the supernatant.

8. Wash the pellet with PBSI for three times.

9. Suspend the pellet in 40 pL of 2X Laemmli sample buffer.

10. Incubate the sample at 95°C for 5 min (see Note 14).

11. Centrifuge the sample at 7000g for 30 s in a microfuge and collect the supernatant (see Note 15).

1. Remove 4 to 15% gradient "ready to use" gel from plastic, remove the comb, and cut the tape along the black line across the entire gel. Pull out the gel (see Note 16).

2. Set up the electrophoresis unit.

3. Place the gel onto the minigel holder cassette of the electrophoresis unit.

4. Load the 10-well gel with 5 pL of standards or 25 pL of the supernatant protein samples.

5. Add 1X running buffer to the electrophoresis cell.

6. Cover the electrophoresis cell with the cell lid.

7. Connect the electrophoresis unit to a power supply. Set the voltage at 150 V.

8. Run the gel at 150 V until the marker dye has run off the edge of the gel.

9. Remove gel and rinse with 1X transfer buffer precooled in ice (4°C).

10. Set up the electrophoretic transfer cell.

11. Soak fiber pad, filter paper, and nitrocellulose membrane in 1X transfer buffer before placing them in the gel cassette.

12. Place nitrocellulose membrane between gel and anode. Inspect to make sure that no air bubble is trapped between gel and membrane.

13. Connect the electrophoretic transfer unit to a power supply.

14. Place the unit on ice or at 4°C cold room.

3.3.3. Western Blotting

1. Remove the nitrocellulose membrane from the electrophoretic transfer unit.

2. Immerse the membrane in PBS containing 5% nonfat milk and gently rock it on a rocking platform at RT for 1 h.

3. Remove the PBS milk solution and add primary antibodies at appropriate dilutions. Incubate at RT for 1 h.

4. Wash the membrane in PBST solution at RT for 5 min. Repeat three times.

5. Incubate membrane in horseradish peroxidase-conjugated secondary antibodies at RT for 1 h.

6. Wash the membrane in PBST at RT for 5 min. Repeat three times.

7. Rinse the membrane.

8. Mix an equal volume of stable peroxide and luminol/enhancer solutions provided in the SuperSignal® West Pico kit.

9. Add the above solutions to the membrane and incubate it at RT for 5 min on a rocking platform.

10. Remove the membrane, blot it quickly with paper towel, and place it between two plastic sheets that have been cut to fit the size of the X-ray film cassette.

11. In a dark room, place the membrane in an X-ray film cassette containing a Kodak X-ray film. Expose the film to X-ray for a suitable time period, usually 5 to 10 s.

12. View the protein bands on X-ray film and analyze their intensities by densitometry.

4. Notes

1. We design oligonucleotides based on the promoter sequences containing the enhancer element(s) to be tested. The length of the oligonucleotide may be a short 20-mer containing a single binding site or as long as 500 bp containing multiple enhancer elements. The sequence is sent to Integrated DNA Technologies (IDT). 5'-biotinylated sense and antisense single-stranded oligonucleotides are synthesized and the sequence is verified by IDT. The oligonucleotides are lyophilized and delivered to our laboratory in powder form. The turnaround time is generally reasonably short.

2. We select affinity-purified rabbit polyclonal antibodies (IgG) specific for transcription factors to be investigated and affinity-purified rabbit nonimmune IgG as control whenever available. The concentrations (dilutions) of specific primary antibodies used in our experiments are generally prepared according to the manufacturers' recommendation.

3. We use goat horseradish peroxidase-conjugated IgG that recognizes rabbit IgG as the secondary antibody when the primary antibody is prepared from rabbit serum.

4. The chemiluminescent kit comes with two separate bottles of reagents: stable peroxide and luminol/enhancer.

5. For convenience, we use plastic sheets from C-Line Products. However, there are many alternatives. For example, Saran™ Wrap will do the work.

6. A 50-mL conical tube is used to ensure a maximal yield of cells.

7. In our experience, a 5-s sonication is sufficient to break the cells. Sonication for a longer period may disrupt the nucleus.

8. Nuclear extracts so prepared do not have detectable cytosolic proteins.

9. As 4 ^g of biotinylated double-stranded oligonucleotides are used for each assay, we mix 4 ^g of sense with 4 ^g of antisense oligonucleotides to generate 4 ^g of 5'-biotinylated double-stranded oligonucleotide in each experiment.

10. The streptavidin-agarose suspension is thick and difficult to mix well. The method described here provides a good mix. After gently stirring with a vortex, the suspension is transferred into a 1-mL tuberculin syringe and mixed; then 2 drops of the mixed suspension are added to the mixture containing nuclear extract proteins and biotinylated oligonucleotides.

11. Each drop contains about 20 ^L of streptavidin-agarose suspension.

12. The incubation time may be shortened to 1 h but should not be shorter than 1 h.

13. It is important to centrifuge the complex at a low speed. A higher speed of centri-fugation may disrupt the complex.

14. This step causes dissociation of transcription factors from the complex.

15. A short spin in a microfuge is sufficient to remove the complex.

16. We have found the Bio-Rad minigel to be convenient. However, minigels from other manufacturers may be used.


The author wishes to thank Dr. Ying Zhu for his contribution to the development of this assay and Drs. Katarzyna Cieslik and Wu-Guo Deng for applying the assay to the study of pharmacologic and pathophysiologic control of transactivator and cotransactivator binding to cyclooxygenase-2 and inducible nitric oxide synthase promoters. This work was supported by grants from the NIH (R01 HL-50675 and P50-NS-23327).


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