which have a net negative charge under the experimental conditions will migrate through the gel on their own. If the protein
is stably bound to nucleic acid, it is more likely to migrate
through the gel due to the negative charge of nucleic acids.
Since many nucleic acid binding proteins have a net positive
charge at physiological pH, they are less likely to run far into
gel on their own.
29. The mobility of the Orange G dye front will depend on the
percentage of the polyacrylamide gel. In our experience, the
Orange G dye front migrates similarly to a ten-nucleotide
single-stranded nucleic acid in an 8% polyacrylamide gel.
Orange G is often preferred as other commonly used dyes
may co-migrate with the nucleic acid and lead to shadows in
imaging.
30. Colorimetric stains such as methylene blue or crystal violet
require no special equipment, but have low sensitivity (and
thus more nucleic acid and protein will be required for the
assay) and require a destaining step. Fluorescent stains such as
ethidium bromide or SYBR Safe (Invitrogen) are often mutagenic and require UV excitation, but have greater sensitivity
and so are often preferred if the nucleic acid is not directly
labeled.
The alternative detection method is to directly label the
nucleic acid. Nucleic acids can be chemically synthesized with a
fluorescent label such as 6-FAM or Alexa dyes, or unlabeled
nucleic acids can be labeled with in-house protocols or commercially available kits. Fluorescent labels are sensitive and their
fluorescence is directly proportional to molarity, allowing semiquantitative analysis of interactions. Alternatively, nucleic acids
can be directly labeled with radioisotopes such as
32
P. Radioactive labeling is advantageous in that it does not
introduce artificial structures that influence binding and is the
most sensitive. However, the use of radioactive labeling
requires training and precautions for radioactive safety.
31. The fragility of the gel will depend on the percentage of acrylamide used. The lower the acrylamide percentage, the more
fragile the gel. To minimize the chance of gel tearing, the tools
and glass plates used to manipulate the gels should be kept wet
by deionized water at all times. If the gel is too fragile, it can
also be stained and directly imaged on the glass plate with a
suitable holder, but the background noise from the glass plate
itself will likely be higher, potentially hindering further analysis.
32. Even if equilibrium has been attained in the binding reaction,
this will be perturbed when applied to an electric field (i.e.,
during electrophoresis). In our hands, EMSAs often lead to an
underestimation of the true affinity, since complexes can
336
James A. W. Stowell et al.
is stably bound to nucleic acid, it is more likely to migrate
through the gel due to the negative charge of nucleic acids.
Since many nucleic acid binding proteins have a net positive
charge at physiological pH, they are less likely to run far into
gel on their own.
29. The mobility of the Orange G dye front will depend on the
percentage of the polyacrylamide gel. In our experience, the
Orange G dye front migrates similarly to a ten-nucleotide
single-stranded nucleic acid in an 8% polyacrylamide gel.
Orange G is often preferred as other commonly used dyes
may co-migrate with the nucleic acid and lead to shadows in
imaging.
30. Colorimetric stains such as methylene blue or crystal violet
require no special equipment, but have low sensitivity (and
thus more nucleic acid and protein will be required for the
assay) and require a destaining step. Fluorescent stains such as
ethidium bromide or SYBR Safe (Invitrogen) are often mutagenic and require UV excitation, but have greater sensitivity
and so are often preferred if the nucleic acid is not directly
labeled.
The alternative detection method is to directly label the
nucleic acid. Nucleic acids can be chemically synthesized with a
fluorescent label such as 6-FAM or Alexa dyes, or unlabeled
nucleic acids can be labeled with in-house protocols or commercially available kits. Fluorescent labels are sensitive and their
fluorescence is directly proportional to molarity, allowing semiquantitative analysis of interactions. Alternatively, nucleic acids
can be directly labeled with radioisotopes such as
32
P. Radioactive labeling is advantageous in that it does not
introduce artificial structures that influence binding and is the
most sensitive. However, the use of radioactive labeling
requires training and precautions for radioactive safety.
31. The fragility of the gel will depend on the percentage of acrylamide used. The lower the acrylamide percentage, the more
fragile the gel. To minimize the chance of gel tearing, the tools
and glass plates used to manipulate the gels should be kept wet
by deionized water at all times. If the gel is too fragile, it can
also be stained and directly imaged on the glass plate with a
suitable holder, but the background noise from the glass plate
itself will likely be higher, potentially hindering further analysis.
32. Even if equilibrium has been attained in the binding reaction,
this will be perturbed when applied to an electric field (i.e.,
during electrophoresis). In our hands, EMSAs often lead to an
underestimation of the true affinity, since complexes can
336
James A. W. Stowell et al.