Electrophoretic Mobility Shift Assay (EMSA) for the detection of nuclear NF-kB

Description of this procedure:

Nuclear extracts
For the preparation of nuclear extracts the cells are gently lysed in a NP-40 containing sucrose buffer while the nuclei remain intact. After a washing step, the nuclei are suspended in a hypotonic "low salt buffer": the nuclei swell. Then a "high salt buffer" is added slowly: the nucleoplasm is extracted into the buffer while the nulear envelop stays intact and retains the genomic DNA. The extract is separated from the nuclear envelop / DNA by centrifugation.
EMSA
For the EMSA, nuclear extracts are incubated with a radioactive labeled oligonucleotide probe which contains the specific recognition sequence for NF-kB. The binding reaction occurs under specific salt/pH conditions in a binding buffer. Poly-dIdC is added to prevent unspecific binding of proteins to the NF-kB oligonucleotide probe and by this to reduce background. After binding, the samples are separated on a non-denaturating PAGE gel and bands are detected by autoradiography. If NF-kB subunits are present in the nuclear extracts, their interaction with the probe shifts the band of the labeled probe up since the complex of probe and NF-kB subunit has a lower electophoretic mobility than the probe alone. The intensity of the shifted band is a measure for the amount of NF-kB in the nuclear fraction (since the probe is added in high excess). To check if the observed shifted bands are specific for NF-kB, competition tests are run: to a protein extract that displays intense shifted bands, additionally to the labeled NF-kB probe a nonlabeled ("cold") oligonucleotide is added in excess. Cold competition oligonucleotides are used that either contain the wildtype NF-kB recognition site or a slightly "mutated" NF-kB site. If the observed signals are NF-kB specific, those signals should disappear in the presence of the cold NF-kB wildtype competitor but should be unaffected in the presence of the cold "mutant" competitor.
Supershift
In order to determine which NF-kB subunits are responsible for the observed shifted bands, the EMSA signals can be "supershifted" by adding different NF-kB subunit-specific antibodies to the binding reactions. The antibodies will bind to the corresponding NF-kB subunit what results in a NF-kB/probe/antibody ternary complex: the electrophoretic mobility of this complex is even lower than that of the NF-kB/probe complex so that a supershifted band can be observed. Sometimes an antibody does not result in a shifted band even if its corresponding NF-kB subunit is present: instead, by binding to its NF-kB subunit the antibody prevents the association of the NF-kB subunit and the labeled probe so that the intensity of the primary shifted signal decreases.


PROCEDURE

Preparation of nuclear extracts

  1. Harvest cells and resuspend them in 1 ml PBS.
  2. Transfer cells into 1,5 ml microfuge tubes and spin down for 5 min at 500xg (2500 rpm in Eppendorf microfuge) at 4 ºC.
  3. Aspirate PBS and resuspend cells in 100 l Sucrose Buffer per 1E+07 cells. Mix by gently pipetting up and down with blue tip.
  4. Spin nuclei down for 5 min at 500g at 4 ºC.
  5. Aspirate off supernatant or, if you need this cytoplasmic fraction, transfer it into new tube.
  6. Wash nuclear pellet with 1 ml Sucrose Buffer (without NP-40). Mix by gently pipetting up and down with blue tip.
  7. Spin nuclei down for 5 min at 500g at 4 ºC.
  8. Aspirate off all the wash buffer.
  9. Resuspend cells in appropriate volume of Low Salt Buffer (e.g. 30 l per 1E+07 cells) by finger vortexing. Be sure that all liquid remains at the bottom of the tube.
  10. Add equal volume High Salt Buffer very slowly (possibly in small aliquots) while mixing with the pipet tip.
  11. Incubate samples for 30-45 min at 4 ºC on a shaker or rotator.
  12. Spin down at 14000g for 15 min (4 ºC).
  13. Transfer supernatant ( = nuclear extract) to new tube
  14. Quantitate protein content of the extracts and store nuclear extracts at -70 ºC. The extracts are ready for use in the EMSA.

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
NFkB wt (anti) : gCC Tgg gAA AgT CCC CTC AAC T

For preparing labeled probe, label both the sense and anti-sense oligo according to following protocol in a total volume of 20 l:

  • 0.5 l of oligonucleotide (200 ng/l)
  • 2 l of 10x PNK reaction buffer
  • 0.5 l of T4 Polynucleotide Kinase
  • 1.5 l of gamma-32P-ATP (if older batch then take 3 l)
  • x l ddH2O.
Incubate for 1 h at 37 ºC.

gamma-32P-ATP is at a concentration of 10 mCi/ml = 370 MBq/ml with 3000 Ci/mmol (corresponds to 3,3 M ATP content).

After labeling, labeled oligonucleotides are purified using the QIAquick Nucleotide Removal Kit (Quiagen). Oligos are eluted from the column with 100 l 10 mM Tris, pH8.0. The purified sense and anti-sense oligos (both 100 l) 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 l but can be increased to 20 l 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.
6 l ddH2O + 2 l Binding Buffer + 1 l dye + 1 l labeled probe.

Tubes No. 2 through N contain the extracts of interest and labeled probe as follows (remember: usual total volume = 10 l):

  1. x l of ddH2O
  2. 1 l of poly-dIdC (1 g/l in TE)
  3. 2 l of Binding Buffer (5X)
  4. x l of Nuclear extract (5 g protein)
  5. 1 l labeled probe (1:15 dilution of kinase reaction)

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 l of "cold" competition probe (20 ng/l stock) to the binding reaction mixture before adding the labeled probe. Adjust amount of water to remain the total volume of 10 l.
The sequence of the wildtype competition probe is the same as the labeled NF-kB specific probe, while the "mutant" probe has a nuclelotide exchange within the recognition region (NFkB binding site underlined with mutated site bold):

NFkB mutant (sense): AgT TgA ggC gAC TTT CCC Agg C
NFkB mutant (anti) : gCC Tgg gAA AgT CgC CTC AAC T

In case of a supershift add 1 l of 1 g/l 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:

  • 8.3 ml of 30% PA solution (29:1)
  • 2,5 ml of 10x TBE
  • 39,2 ml ddH2O
  • 50 l TEMED
  • 0,5 ml APS (10%)
Load the EMSA samples into the wells of the gel. Run the gel for 2,5 hours in 0,5x TBE at 1 mA/cm (at the start the voltage is about 120 V, during the course of electrophoresis the voltage increases to > 200 V). After PAGE, transfer the gel to a double layer of Whatman paper and dry the gel on a gel dryer for 45 - 60 min at 75 ºC. Expose the dried gel to autoradiography film (at -70 ºC with enhancer foils).


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 l of 1 M CaCl2
100 l of 1 M MgOAc
10 l 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 l of 1M MgCl2
0.4 ml of 2.5 M KCl
20 l 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 g/ml leupeptin, aprotinin, pepstatin
10 ml of 100 mM HEPES (pH 7.9)
75 l of 1M MgCl2
16 ml of 2.5 M KCl
20 l 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 l of 0.5 M EDTA (pH 8.0)
50 l Triton-X 100
7.87 g glycerol
add DTT fresh before use