4. Ammonium persulfate (APS): 10% (w/v) solution. Weigh 5 g
ammonium persulfate. Dissolve in MilliQ water and top up to
50 ml. Freeze aliquots at À20
C (see Note 4).
5. N,N,N,N
0 -Tetramethyl-ethylenediamine (TEMED): Supplied
as a liquid. Can be stored at room temperature, or at 4
C to
reduce vapor.
6. 1Â TBE running buffer: Dilute the 10Â TBE stock tenfold in
MilliQ water.
7. 10Â EMSA loading dye: 0.025% (w/v) Orange G and 20%
(v/v) glycerol (see Note 5).
8. Electrophoresis chamber: Use a vertical electrophoresis apparatus with corresponding glass plates, spacers, and wellforming combs. Clamps and/or a gel-casting stand are
required for gel preparation. This protocol uses the MiniPROTEAN Tetra cell with 1.0 mm thick backing plates and
combs (Bio-Rad) (see Note 6).
9. Silanization solution: 5% (v/v) dimethyldichlorosilane
(!99.5%; Sigma-Aldrich catalog no.: 440272) or chlorotrimethylsilane (!99.5%; Sigma-Aldrich catalog no.: 386529) in
heptane if silanization of glassware is carried out (see Note 7).
10. Power supply: Minimum 100 V, 25 mA capacity.
11. Tapered or round gel-loading pipette tips are useful but not
essential.
2.2 Protein
and Nucleic Acid
Preparation
The exact requirements for reagents will depend on the properties
of the proteins and nucleic acids to be studied. Optimal conditions
for each EMSA experiment must therefore be determined for every
study. Here, we focus on the general requirements of each sample
and the “standard” conditions that we have outlined in the
methods.
1. Protein: The protein sample should be highly pure (ideally
>95% as assayed by SDS-PAGE) and in a buffer with conditions where the protein is known to be stable (usually around
pH 7–8). The protein should either be freshly purified or flash
frozen and stored at À80
C (see Note 8).
2. TE buffer: 10 mM Tris pH 8.0, 0.5 mM EDTA. For long-term
storage of nucleic acids, see Note 9.
3. DEPC-treated water: Treat MilliQ water with 0.1% (v/v)
DEPC for at least 2 h at 37
C. Autoclave the resulting solution
(121
C, 15 min) to inactivate trace DEPC.
4. DNA or RNA preparation: DNA samples should be highly
purified and stored in either MilliQ water or TE buffer (see
Note 9). Because of the relatively low cost and commercial
availability, we use chemically synthesized DNA. RNA samples
324
James A. W. Stowell et al.
ammonium persulfate. Dissolve in MilliQ water and top up to
50 ml. Freeze aliquots at À20
C (see Note 4).
5. N,N,N,N
0 -Tetramethyl-ethylenediamine (TEMED): Supplied
as a liquid. Can be stored at room temperature, or at 4
C to
reduce vapor.
6. 1Â TBE running buffer: Dilute the 10Â TBE stock tenfold in
MilliQ water.
7. 10Â EMSA loading dye: 0.025% (w/v) Orange G and 20%
(v/v) glycerol (see Note 5).
8. Electrophoresis chamber: Use a vertical electrophoresis apparatus with corresponding glass plates, spacers, and wellforming combs. Clamps and/or a gel-casting stand are
required for gel preparation. This protocol uses the MiniPROTEAN Tetra cell with 1.0 mm thick backing plates and
combs (Bio-Rad) (see Note 6).
9. Silanization solution: 5% (v/v) dimethyldichlorosilane
(!99.5%; Sigma-Aldrich catalog no.: 440272) or chlorotrimethylsilane (!99.5%; Sigma-Aldrich catalog no.: 386529) in
heptane if silanization of glassware is carried out (see Note 7).
10. Power supply: Minimum 100 V, 25 mA capacity.
11. Tapered or round gel-loading pipette tips are useful but not
essential.
2.2 Protein
and Nucleic Acid
Preparation
The exact requirements for reagents will depend on the properties
of the proteins and nucleic acids to be studied. Optimal conditions
for each EMSA experiment must therefore be determined for every
study. Here, we focus on the general requirements of each sample
and the “standard” conditions that we have outlined in the
methods.
1. Protein: The protein sample should be highly pure (ideally
>95% as assayed by SDS-PAGE) and in a buffer with conditions where the protein is known to be stable (usually around
pH 7–8). The protein should either be freshly purified or flash
frozen and stored at À80
C (see Note 8).
2. TE buffer: 10 mM Tris pH 8.0, 0.5 mM EDTA. For long-term
storage of nucleic acids, see Note 9.
3. DEPC-treated water: Treat MilliQ water with 0.1% (v/v)
DEPC for at least 2 h at 37
C. Autoclave the resulting solution
(121
C, 15 min) to inactivate trace DEPC.
4. DNA or RNA preparation: DNA samples should be highly
purified and stored in either MilliQ water or TE buffer (see
Note 9). Because of the relatively low cost and commercial
availability, we use chemically synthesized DNA. RNA samples
324
James A. W. Stowell et al.