EMSA for NFkB Binding Activity

The procedures for the binding reaction include the following: incubate the nuclear extracts with [y-32P]ATP-labeled oligonucleotide probe that contains the specific binding sequence for NF-kB (the binding reaction occurs under specific salt/pH conditions in a binding buffer). Add Poly-dIdC into the binding reaction mixtures to prevent nonspecific binding of proteins to the NF-kB oligonucleotide probe. Determine the subunits of NF-kB binding activity in the nuclear extracts by competition assay and antibody supershift analysis. Separate the binding reaction mixtures on a nondenaturing PAGE gel. Finally, dry the PAGE gel and detect the binding bands by autoradiography (17-20).

3.2.1. Labeling of Consensus Oligonucleotide With [y-32P]ATP (see Notes 2 and 3)

1. Take a sterile 1.5-mL Eppendorf tube and add the items sequentially as shown in Table 1.

2. Incubate the reaction mixture at 37°C for 10 min.

3. Stop the reaction by addition of 1 pL of 0.5 M EDTA.

3.2.2. Determination of the Percent of [y-32P]ATP Incorporation Into the Oligonucleotide

1. Take 1 pL of the labeled oligonucleotide and spot onto a DE81 filter paper.

2. Dry the DE81 paper briefly under a heat lamp.

3. Place the DE1 paper into a vial and count cpm in a scintillation counter.

4. After counting, wash the DE81 paper in 50 mL of 0.5 M Na2HPO4, twice for 5 min each, to remove the unincorporated [y-32P]ATP. Dry the washed DE81 filter paper under a heat lamp.

5. Place the DE81 paper into a vial and count the cpm in a scintillation counter.

6. Calculate the percent incorporation of [y-32P]ATP into the oligonucleotide.

Percent incorporation = cpm incorporated/total cpm X 100%

3.2.3. Binding Reaction

1. Take a set of 0.5-mL Eppendorf tubes and mark them with designated numbers using a marker pen.

2. Add each the following items sequentially as shown in Table 2 into designated 0.5-mL Eppendorf tubes in a total 10 pL of reaction volume (see Note 4).

Nuclear extracts x pL (5-10 pg)

5X binding buffer 2 pL

Table 1

Labeling NF-kB Oligonucleotides With [y-32P]ATP

Items Volume

NF-kB oligonucleotide (1.75 pmol/(L) 2 mL

T4 polynucleotide kinase 10X buffer 1 mL

dH2O 1 mL

T4 polynucleotide kinase (5-10 U/(L) 1 mL

Total reaction volume 10 mL

Table 2

Binding Reaction Mixtures

Sample No

Nuclear proteins 5X binding Buf Buf C

P-oligo probe

Negative control 0 2 pL 7 pL 1 pL

32P-oligonucleotide probe 1 (L

3. Incubate the reaction mixtures at room temperature for 20 min.

4. Stop the binding reaction by adding 2 (L of DNA loading buffer.

5. Briefly centrifuge a microcentrifuge.

6. Load the samples on 5% nondenatured PAGE gel.

3.2.4. Electrophoresis

1. Set up the polyacrylamide gel casting: take clean plates and the spacers. Tighten them to make the casting module and fit it on the stand with the rubber spacer on bottom.

2. Preparation of 5% nondenaturing polyacrylamide gel (40 mL) as below (see Note 5).

Components 5% gel

TBE 5X buffer 8.00 mL

30:0.8 (acrylamide:Bis-acrylamide) 6.70 mL

80% glycerol 1.25 mL

dH2O 23.73 mL

TEMED 20 (L

10% Ammonium persulfate 300 (L

3. Use a 20-mL syringe to pour the gel on the side of cast near spacers. Fill the cast from bottom without any air bubbles inside. Take the comb and insert it into the gel without trapping any air bubbles. Polymerize the gel for 0.5 h. Make sure its not leaking when leaving it to polymerize.

Fig. 1. Lipopolysaccharide (LPS) stimulates nuclear factor-kB (NF-kB) binding activity in mast cells. Mast cells were stimulated with or without LPS at 100 ng/mL for 30 and 60 min, respectively, and nuclear proteins were isolated from the cells. Five micrograms of nuclear proteins from each sample were used for electrophoretic mobility shift assay (EMSA) of NF-kB binding activity. NF-kB binding activity is labeled on the right; nb, nonspecific binding.

4. Prerun the gel for 20 min at 100 V before loading the samples.

5. Load the reaction samples on the gel and run the get at room temperature, in 0.5X TBE buffer at 100 V for approx 2 h or until the bromophenol blue dye is just out of the gel.

6. Collect the gel running buffer into a radioactive liquid waste container. Take the gel cast out and separate the glass plate carefully.

7. Place the gel on a sheet of plastic wrap and cover the gel with Whatman 3MM filter papers and lift the gel. Wrap the gel with plastic wrap and dry with a gel-drier at 80°C for 1 h.

8. Expose the dried gel to X-ray film several hours to overnight at -80°C with an intensifying screen.

9. Develop the film in a dark room (see Note 6).

10. Scan the gel to determine the relative integrated intensity of the bands. Fig. 1 shows NF-kB activation in HMC-1 mast cells stimulated with LPS (100 ng/mL) for 30 and 60 min.

3.2.5. Competition and Supershift Assay

To determine the specificity of NF-kB binding activity and the subunits of activated NF-kB, competition with unlabeled oligonucleotides and supershift assay with specific antibodies against subunits of NF-kB are performed as in the next list.

1. Take a set of 0.5-mL Eppendorf tubes and mark them with designated numbers using a marker pen.

2. Add each of the following items sequentially as shown in Table 3 into designated 0.5-mL Eppendorf tubes in a total 10 ^L of reaction volume.

5X binding buffer 2 (L

Unlabeled NF-kB oligonucleotide 2 (L

Other oligonucleotide 2 (L

32P-oligonucleotide probe 1 (L

3. Incubate the binding reaction mixture for 20 min at room temperature.

5. Incubate the binding reaction mixture for 1-2 h at 4°C.

6. Stop the binding reaction by addition of 2 (L of DNA loading buffer.

7. Briefly centrifuge in a microcentrifuge.

8. Load the samples on a 5% nondenatured PAGE gel.

Figure 2 shows specificity of NF-kB binding activity determined by addition of unlabeled oligonucleotides and by an antibody supershift assay.

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