18. Reagents such as protein and nucleic acids are normally added
from stocks at 10Â concentration to minimize the contribution of buffer carryover to the EMSA reaction. If greater
volumes of reagent are added, the 10Â EMSA buffer can be
adjusted accordingly to account for buffer carryover.
19. The nucleic acid concentration should be optimized depending on detection of the nucleic acid and the experiment type.
For fluorescently labeled nucleic acids and detection by a laser
scanner such as the Typhoon FLA Scanner (GE), approximately 0.01 pmol fluorophore suffices for detection. Furthermore, the amount of nucleic acid should not exceed the
maximum detection limit (for example, pixel saturation) on
the detector. Many nucleic acid:protein interactions have K d s
less than 10 nM. In this scenario, fluorescently labeled RNAs
are not optimal substrates for EMSAs because of ligand depletion (see Note 33). Instead, sub-picomolar radiolabeled
nucleic acid substrates are preferred.
20. Protein concentration should be accurately determined, for
example, by measuring the absorbance at 280 nm (A 280 ) and
calculating the protein concentration using a theoretical
extinction coefficient. Measurements should be made under
denaturing conditions and compared with the native protein
to see if there is a substantial difference in the calculated
concentration. If possible, a UV spectrum of the sample should
be taken. Absorbance in the region beyond which proteins
normally absorb (>320 nm) indicates the presence of light
scattering due to aggregation or particulates, which would
lead to overestimation of the absorbance at 280 nm due to
unaggregated protein and hence its concentration. Samples
with significant absorbance at 320–340 nm region should be
centrifuged to remove any aggregates. If the sample does not
contain any tryptophan residues, which are the main source of
protein absorbance at 280 nm, consider using a colorimetric
assay such as the Bradford method.
21. It is preferable to have a protein stock at a high concentration
(>100 μM) as long as the protein does not aggregate. This is
because a greater concentration range can be sampled and the
A 280 measurement is more accurate (see Note 20).
22. Protein dilution buffer composition is sample dependent (see
Note 8). To minimize carryover of buffer components into the
binding reaction, our default dilution buffer contains 20 mM
HEPES pH 7.5, 100 mM NaCl, and 0.5 mM TCEP.
23. To minimize the adsorption of protein to plastic tubes, we use
low-binding plasticware such as protein Lo-Bind tubes
(Eppendorf). This also ensures that the concentration of the
protein is consistent in the assays.
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James A. W. Stowell et al.
from stocks at 10Â concentration to minimize the contribution of buffer carryover to the EMSA reaction. If greater
volumes of reagent are added, the 10Â EMSA buffer can be
adjusted accordingly to account for buffer carryover.
19. The nucleic acid concentration should be optimized depending on detection of the nucleic acid and the experiment type.
For fluorescently labeled nucleic acids and detection by a laser
scanner such as the Typhoon FLA Scanner (GE), approximately 0.01 pmol fluorophore suffices for detection. Furthermore, the amount of nucleic acid should not exceed the
maximum detection limit (for example, pixel saturation) on
the detector. Many nucleic acid:protein interactions have K d s
less than 10 nM. In this scenario, fluorescently labeled RNAs
are not optimal substrates for EMSAs because of ligand depletion (see Note 33). Instead, sub-picomolar radiolabeled
nucleic acid substrates are preferred.
20. Protein concentration should be accurately determined, for
example, by measuring the absorbance at 280 nm (A 280 ) and
calculating the protein concentration using a theoretical
extinction coefficient. Measurements should be made under
denaturing conditions and compared with the native protein
to see if there is a substantial difference in the calculated
concentration. If possible, a UV spectrum of the sample should
be taken. Absorbance in the region beyond which proteins
normally absorb (>320 nm) indicates the presence of light
scattering due to aggregation or particulates, which would
lead to overestimation of the absorbance at 280 nm due to
unaggregated protein and hence its concentration. Samples
with significant absorbance at 320–340 nm region should be
centrifuged to remove any aggregates. If the sample does not
contain any tryptophan residues, which are the main source of
protein absorbance at 280 nm, consider using a colorimetric
assay such as the Bradford method.
21. It is preferable to have a protein stock at a high concentration
(>100 μM) as long as the protein does not aggregate. This is
because a greater concentration range can be sampled and the
A 280 measurement is more accurate (see Note 20).
22. Protein dilution buffer composition is sample dependent (see
Note 8). To minimize carryover of buffer components into the
binding reaction, our default dilution buffer contains 20 mM
HEPES pH 7.5, 100 mM NaCl, and 0.5 mM TCEP.
23. To minimize the adsorption of protein to plastic tubes, we use
low-binding plasticware such as protein Lo-Bind tubes
(Eppendorf). This also ensures that the concentration of the
protein is consistent in the assays.
334
James A. W. Stowell et al.