Electrophoretic mobility shift assays (EMSAs) are used to investigate DNA-protein interactions. Non-denaturing conditions are chosen for electrophoresis to ensure naïve conformation of the proteins. Protein-bound DNA is migrating slower than free DNA visible by a band shift after radiography. In the present work, EMSAs were used to investigate the DNA-binding ability of the CREB mutant CREB-K290E. Furthermore the occurrence of both CREB and TORC1 in complex with the CRE of the rat somatostatin gene promoter was investigated by supershift experiments (11.d.II).
11.a Buffers and solutions
TBE 5x 2 L
Tris 450 mM 108.9 g
Boric acid 450 mM 55.6 g
EDTA pH 8.0 10 mM 40 mL 0.5 M EDTA
Acrylamide for Shift-Gel 40% (w/v) 100 mL
Acrylamide 38% (w/v) 38 g
Bis-acrylamide 2% (w/v) 2 g
Stop mix blue for EMSA 10 mL
Glycerol 30%(v/v) 3.39 mL 87% Glycerol
Bromophenolblue 0.25% (w/v) 0.025 g Xylene cyanol FF 0.25% (w/v) 0.025 g
H2O ad 10 mL
Binding buffer 500 µL
Tris / HCl pH 7.5 50 mM 25 µL 1 M Tris/HCl pH 7.5
NaCl 100 mM 50 µL 1 M NaCl
Glycerin 10% (v/v) 100 µL 50% Glycerin
MgCl2 20 mM 10 µL of 1 M MgCl2-Sol.
BSA 12 µg / mL 6 µL of 1 mg / mL BSA-Sol.
poly(dI-dC) 80 mg / mL 40 µL of 1 µg /µL stocksol.
All buffers were stored at room temperature. The acrylamide solution was stored at 4°C.
BSA and poly(dI-dC) were added freshly before use of the binding buffer.
11.b Gel preparation
For EMSAs non-denaturing gels were used which are free from SDS.
For a 5% polyacrylamide gel the following components were mixed:
Acrylamide 5% (w/w) 6.25 mL of acrylamide for shift gels (40%)
TBE 0.5 x 5 mL 5 x TBE
H2O 38 mL
TEMED 50 µL
APS 500 µL 10% APS
The gel solution was prepared, cast between 2 glass electrophoresis plates (18 cm x 16 cm x 3 mm), and the comb forming the slots was inserted immediately. After polymerization the gel was placed in the electrophoresis chamber SE 600. The chamber was filled with 0.5 x TBE buffer and a pre-electrophoresis was performed for 1 h at 180 V to prewarm the gel.
11.c Labeling of oligonucleotides
11.c.I. Annealing
To generate double-stranded probes, complementary oligonucleotides were used. The oligonucleotides were designed such, that the probe contains a 5’-GATC overhang after annealing (Oetjen et al., 1994). For the reaction the following components were mixed in a 0.5 mL tube:
Oligonucleotide forward 100 pmol Oligonucleotide reverse 100 pmol
NaCl 10 mM
H2O ad 50 µL
The tubes were placed in water of 85°C temperature which was cooled down to room temperature overnight.
11.c.II. Labeling with [32P]
The probes were labeled by a fill-in reaction using [α-32P]-dCTP (3000 Ci/mmol, Hartmann Analytics) and Klenow enzyme. For the reaction the following components were used:
Double-stranded probe 10 pmol
dATP 1 mM
dGTP 1 mM
dTTP 1 mM
[α-32P]-dCTP 20 µCi
Klenow buffer 1 x
Klenow fragment 2 U
H2O ad 20 µL
The reaction was performed for 30 min at 25°C (Eppendorf thermomixer).
11.c.III. Purification of the labeled probe
The labeled probe was purified from the free [α-32P]-dCTP by gel filtration chromatography using Mini Quick Spin™ Oligo Columns (Roche) according to the manufacturer’s instruction. The Sephadex matrix was homogenized by vortexing and flipping. The storage buffer inside the column was removed by centrifugation for 1 min at 1,000xg (Biofuge 15R) at 4°C. The column was placed in a new 1.5 mL tube and the probe was carefully pipetted on the matrix. The purification was performed by centrifugation for 1 min at 1,000xg (Biofuge 15R) at 4°C. The eluate contained the purified probe which was quantified immediately.
11.c.IV. Quantification of the incorporation
1 µL of the probe was mixed with 4 mL of scintillation suspension and placed in a beta counter (LS1801, Beckmann). Depending on the counts per min (cpm) detected, reflecting the quality of incorporation of [α-32P]-dCTP, the probe was diluted to a concentration of 20,000 cpm per µL. The probe was stored at -20°C.
11.d Binding reaction
11.d.I. General procedure
In the present study, GST-fusion proteins of CREB wild-type and mutant K290E were analyzed with respect to their ability to bind to the CRE of the rat somatostatin gene. The reaction was performed with some modification as described before (Knepel et al., 1990).
Defined amounts of proteins were incubated in binding buffer for 10 min on ice. The binding buffer contains poly(deoxyinosinic-deoxycytidylic) acid [poly(dI-dC)], which is a polymer acting as unspecific competitor DNA. Afterwards the amount of labeled probe corresponding to 20,000 cpm was added and incubated on ice for 15 min. Subsequently, the desired amount of stop mix was added and the samples were loaded on the gel.
11.d.II. Supershift
To verify the presence of a certain protein in a protein-DNA complex, the proteins are incubated with a specific antibody against the protein. Since the electrophoresis is performed under non-denaturing conditions, the protein-DNA complex is expected to migrate differently when an antibody is bound to the protein. In the present work, the CREB-KID antibody was used to detect GST-CREB proteins in complex with DNA,
whereas the panTORC antibody was employed to detect TORC. The respective amounts of antibody are given in table 9 (3.f). To perform the supershift, the proteins were incubated for 20 min at room temperature in binding buffer without poly(dI-dC) but containing the respective amounts of antibody. Afterwards the mixture was stored on ice at 4°C. The next day, poly(dI-dC) and the labeled probe were added and left for 15 min on ice. The samples were loaded immediately with sufficient amount of stop mix and electrophoresed.
11.e Electrophoresis
The slots of the gel were washed carefully with TBE buffer. The samples were loaded on the gel and a constant power of 180 V was applied to the gel (Biometra® Standard Power Pack P25). Electrophoresis was performed until the lowest band from the blue Stop mix had passed ¾ of the gel. Afterwards the gel was dried and analyzed as described (section 6.f).