Electrophoretic Mobility Shift Assay (EMSA) for the detection of nuclear NF-kB |
---|
Description of this procedure:
Nuclear extracts |
PROCEDURE
Preparation of nuclear extracts
EMSA
A. Labeling of the oligonucleotide probe: T4 Polynucleotide Kinase Reaction
For the NF-kB specific probe, we first label the sense- and antisense oligonucleotide separately, then we purify the oligos before annealing them to obtain a 5' and 3' labeled double-stranded DNA probe.
Here are the wildtype sequences of the sense and antisense oligos (with NF-kB binding site sequence underlined):
NFkB wt (sense): AgT TgA ggg gAC TTT CC Cag gC
For preparing labeled probe, label both the sense and anti-sense oligo according to following protocol in a total volume of 20 µl:
gamma-32P-ATP is at a concentration of 10 mCi/ml = 370 MBq/ml with 3000 Ci/mmol (corresponds to 3,3 µM ATP content).
After labeling, labeled oligonucleotides are purified using the QIAquick Nucleotide Removal Kit (Quiagen). Oligos are eluted from the column with 100 µl 10 mM Tris, pH8.0. The purified sense and anti-sense oligos (both 100 µl) are combined and incubated for 10 min at RT for annealing. The probe is now ready for use.
B. Binding Reaction:
Work on ice, except during the final incubation at RT! The total volume of the binding reactions is usually 10 µl but can be increased to 20 µl if extracts are dilute. When setting up the binding reaction, add the components in the order given below.
Tube No. 1 should contain labeled probe alone together with dye (BPB/XC), e.g.
Tubes No. 2 through N contain the extracts of interest and labeled probe as follows (remember: usual total volume = 10 µl):
After addition of all components, spin reaction tubes briefly in microfuge to bring all fluid down to the bottom of the tube. Incubate 30 min at RT. Then load all the samples on the nondenaturating 5% PA gel (see below).
For a competition test add 1 µl of "cold" competition probe (20 ng/µl stock) to the binding reaction mixture before adding the labeled probe. Adjust amount of water to remain the total volume of 10 µl.
NFkB mutant (sense): AgT TgA ggC gAC TTT CCC Agg C
In case of a supershift add 1 µl of 1 µg/µl antibodies (specific for p50, p52, p65 = RelA, RelB or cRel subunit) to the binding reaction mixture before adding the labeled probe.
C. PAGE:
Prepare a 5% non-denaturating PA (0.5 x TBE) gel for running the EMSA samples. The dimensions of the gels are for example 20x17 cm. Use 1,5 mm spacers and a comb with about 15 wells.
For 50 ml of 5% PA gel:
|
Buffers | |
Sucrose Buffer | |
COMPOSITION: | RECIPE for 50 ml: |
0.32 M Sucrose 10 mM Tris HCl pH 8.0 3 mM CaCl2 2 mM MgOAc 0.1 mM EDTA 0.5% NP-40 1 mM DTT 0.5 mM PMSF |
5.47 g sucrose 0.5 ml of 1 M Tris HCl, pH 8.0 150 µl of 1 M CaCl2 100 µl of 1 M MgOAc 10 µl of 0.5 M EDTA (pH 8.0) 2.5 ml of 10% NP-40 42.4 ml H2O add DTT and PMSF fresh before use |
Low Salt Buffer | |
COMPOSITION: | RECIPE for 50 ml: |
20 mM HEPES (pH 7.9) 1.5 mM MgCl2 20 mM KCl 0.2 mM EDTA 25% glycerol (v/v) 0.5 mM DTT 0.5 mM PMSF |
10 ml of 100 mM HEPES (pH 7.9) 75 µl of 1M MgCl2 0.4 ml of 2.5 M KCl 20 µl of 0.5 M EDTA (pH 8.0) 15.75 g glycerol 27 ml H2O add DTT and PMSF fresh before use |
High Salt Buffer | |
COMPOSITION: | RECIPE for 50 ml: |
20 mM HEPES (pH 7.9) 1.5 mM MgCl2 800 mM KCl 0.2 mM EDTA 25% glycerol (v/v) 1% NP-40 0.5 mM DTT 0.5 mM PMSF 4.0 µg/ml leupeptin, aprotinin, pepstatin |
10 ml of 100 mM HEPES (pH 7.9) 75 µl of 1M MgCl2 16 ml of 2.5 M KCl 20 µl of 0.5 M EDTA (pH 8.0) 15.75 g glycerol 5 ml of 10% NP-40 6.4 ml H2O add DTT, PMSF and proteinase inhibitors fresh before use |
5X Binding Buffer | |
COMPOSITION: | RECIPE for 10 ml: |
50 mM Tris HCl (pH 8.0) 750 mM KCl 2.5 mM EDTA 0.5 % Triton-X 100 62.5 % glycerol (v/v) 1 mM DTT |
0.5 ml of 1 M Tris HCl (pH 8.0) 3 ml of 2.5 M KCl 50 µl of 0.5 M EDTA (pH 8.0) 50 µl Triton-X 100 7.87 g glycerol add DTT fresh before use |