acrylamide. Gas-stabilized ultrapure acrylamide:bis-acrylamide
solution (such as National Diagnostics AccuGel) is readily
available from commercial sources. This also minimizes the
risk of degradation products, such as acrylic acid, interfering
with electrophoresis.
4. Ammonium persulfate is unstable but can be stored frozen at
À20
C for 6 months. Alternatively, a small volume of APS can
be made fresh and kept at 4
C for up to a week.
5. A colored loading dye allows sample visualization during pipetting and gel loading and increases the density of the sample,
causing it to sink to bottom of the wells. We routinely use
Orange G since bromophenol blue can migrate at the same
position as free nucleic acid in the gel, interfering with visualization. If the dyes nonspecifically bind proteins or nucleic acids,
samples can be run with a loading buffer containing only
glycerol and without a dye. If glycerol is incompatible with
the sample, Ficoll 400 (~1–2% w/v final concentration) can
be used as an alternative. In the absence of loading dye, a lane
can be kept free for the addition of a dye to monitor migration.
6. The dimensions of gels cast using this system are 8.3 Â 7.3 cm.
For greater resolving power, larger gels can be used. Many
other suitable apparatus options exist, including those that
accommodate precast native PAGE gels.
7. Preparation of the silanization solution and application of the
solution to glassware should be carried out under a fume hood
as fumes from both the silanizing compound and the organic
solvent are toxic.
8. The choice of buffer for the interaction analysis is an important
consideration. Parameters to consider include: salt concentration (ionic strength), pH (accounting for buffer pK a ), additional metal ions, and additives. Protein–nucleic acid
interactions can be dependent on macromolecular charge,
which in turn is sensitive to salt concentration and pH. We
usually use solutions which approximate physiological salt concentrations and pH. Additional metal ions, such as divalent
cations or potassium (for example, in the case of
G-quadruplex formation [18]), may be important for mediating protein–nucleic acid interactions. In some cases, EDTA in
the gel and running buffer can disrupt interactions—if this is
the case, EDTA can be omitted and/or additional magnesium
can be included. Additives such as detergents or reducing
agents may be important to solubilize macromolecules or
maintain them in a near-physiological state. If proteins tend
to adhere to surfaces, a low concentration of surfactant can also
be added. Finally, a polyanionic additive such as tRNA or
heparin can be used (start with 0.01 mg/ml) to reduce nonspecific binding.
332
James A. W. Stowell et al.
solution (such as National Diagnostics AccuGel) is readily
available from commercial sources. This also minimizes the
risk of degradation products, such as acrylic acid, interfering
with electrophoresis.
4. Ammonium persulfate is unstable but can be stored frozen at
À20
C for 6 months. Alternatively, a small volume of APS can
be made fresh and kept at 4
C for up to a week.
5. A colored loading dye allows sample visualization during pipetting and gel loading and increases the density of the sample,
causing it to sink to bottom of the wells. We routinely use
Orange G since bromophenol blue can migrate at the same
position as free nucleic acid in the gel, interfering with visualization. If the dyes nonspecifically bind proteins or nucleic acids,
samples can be run with a loading buffer containing only
glycerol and without a dye. If glycerol is incompatible with
the sample, Ficoll 400 (~1–2% w/v final concentration) can
be used as an alternative. In the absence of loading dye, a lane
can be kept free for the addition of a dye to monitor migration.
6. The dimensions of gels cast using this system are 8.3 Â 7.3 cm.
For greater resolving power, larger gels can be used. Many
other suitable apparatus options exist, including those that
accommodate precast native PAGE gels.
7. Preparation of the silanization solution and application of the
solution to glassware should be carried out under a fume hood
as fumes from both the silanizing compound and the organic
solvent are toxic.
8. The choice of buffer for the interaction analysis is an important
consideration. Parameters to consider include: salt concentration (ionic strength), pH (accounting for buffer pK a ), additional metal ions, and additives. Protein–nucleic acid
interactions can be dependent on macromolecular charge,
which in turn is sensitive to salt concentration and pH. We
usually use solutions which approximate physiological salt concentrations and pH. Additional metal ions, such as divalent
cations or potassium (for example, in the case of
G-quadruplex formation [18]), may be important for mediating protein–nucleic acid interactions. In some cases, EDTA in
the gel and running buffer can disrupt interactions—if this is
the case, EDTA can be omitted and/or additional magnesium
can be included. Additives such as detergents or reducing
agents may be important to solubilize macromolecules or
maintain them in a near-physiological state. If proteins tend
to adhere to surfaces, a low concentration of surfactant can also
be added. Finally, a polyanionic additive such as tRNA or
heparin can be used (start with 0.01 mg/ml) to reduce nonspecific binding.
332
James A. W. Stowell et al.