assay could be performed with three differentially labeled substrates: Alexa Fluor 405, Alexa Fluor 568, and Alexa Fluor 790.
Alternatively, one nucleic acid can be labeled with a fluorophore or radioactive isotope (“hot”), and the other nucleic acid
kept unlabeled (“cold”). This latter protocol can be used if
there is no way of scanning each channel separately.
38. Alternatively, a fixed concentration of P1 and labeled N1 can be
used, and the concentration of nucleic acid N2 can be titrated.
As the concentration of N2 is increased, P1 may be titrated
away from the P1–N1 complex. Labeled N1 can thus be
detected as a free nucleic acid band.
39. The nucleic acid concentration used will depend on the particular experiment. We often perform these assays with relatively
high nucleic acid concentrations to mirror that used in activity
assays. In Fig. 2, the two different concentrations illustrate how
the differences in specific binding depend on both probe and
protein concentration.
Acknowledgments
We thank Michael Webster for providing purified SpPuf3. This
work was supported by a Herchel Smith PhD Studentship from
the University of Cambridge (to T.T.L.T.); the European Union’s
Horizon 2020 research and innovation programme (ERC grant
725685) (to L.A.P.); and Medical Research Council grant
MC_U105192715 (L.A.P.).
References
1. Pagano JM, Clingman CC, Ryder SP (2011)
Quantitative approaches to monitor proteinnucleic acid interactions using fluorescent
probes. RNA 17(1):14–20. https://doi.org/
10.1261/rna.2428111
2. Fried M, Crothers DM (1981) Equilibria and
kinetics of lac repressor-operator interactions
by polyacrylamide gel electrophoresis. Nucleic
Acids Res 9(23):6505–6525
3. Garner MM, Revzin A (1981) A gel electrophoresis method for quantifying the binding of
proteins to specific DNA regions: application
to components of the Escherichia coli lactose
operon regulatory system. Nucleic Acids Res 9
(13):3047–3060
4. Hellman LM, Fried MG (2007) Electrophoretic mobility shift assay (EMSA) for detecting
protein-nucleic acid interactions. Nat Protoc 2
(8):1849–1861. https://doi.org/10.1038/
nprot.2007.249
5. Berger R, Duncan MR, Berman B (1993)
Nonradioactive gel mobility shift assay using
chemiluminescent detection. BioTechniques
15(4):650–652
6. Hsieh YW, Alqadah A, Chuang CF (2016) An
optimized protocol for electrophoretic mobility shift assay using infrared fluorescent
dye-labeled oligonucleotides. J Vis Exp
117:54863. https://doi.org/10.3791/54863
7. Jing D, Agnew J, Patton WF et al (2003) A
sensitive two-color electrophoretic mobility
shift assay for detecting both nucleic acids and
protein in gels. Proteomics 3(7):1172–1180.
https://doi.org/10.1002/pmic.200300438
8. Rye HS, Drees BL, Nelson HC, Glazer AN
(1993) Stable fluorescent dye-DNA complexes
in high sensitivity detection of protein-DNA
interactions. Application to heat shock transcription factor. J Biol Chem 268
(33):25229–25238
338
James A. W. Stowell et al.
Alternatively, one nucleic acid can be labeled with a fluorophore or radioactive isotope (“hot”), and the other nucleic acid
kept unlabeled (“cold”). This latter protocol can be used if
there is no way of scanning each channel separately.
38. Alternatively, a fixed concentration of P1 and labeled N1 can be
used, and the concentration of nucleic acid N2 can be titrated.
As the concentration of N2 is increased, P1 may be titrated
away from the P1–N1 complex. Labeled N1 can thus be
detected as a free nucleic acid band.
39. The nucleic acid concentration used will depend on the particular experiment. We often perform these assays with relatively
high nucleic acid concentrations to mirror that used in activity
assays. In Fig. 2, the two different concentrations illustrate how
the differences in specific binding depend on both probe and
protein concentration.
Acknowledgments
We thank Michael Webster for providing purified SpPuf3. This
work was supported by a Herchel Smith PhD Studentship from
the University of Cambridge (to T.T.L.T.); the European Union’s
Horizon 2020 research and innovation programme (ERC grant
725685) (to L.A.P.); and Medical Research Council grant
MC_U105192715 (L.A.P.).
References
1. Pagano JM, Clingman CC, Ryder SP (2011)
Quantitative approaches to monitor proteinnucleic acid interactions using fluorescent
probes. RNA 17(1):14–20. https://doi.org/
10.1261/rna.2428111
2. Fried M, Crothers DM (1981) Equilibria and
kinetics of lac repressor-operator interactions
by polyacrylamide gel electrophoresis. Nucleic
Acids Res 9(23):6505–6525
3. Garner MM, Revzin A (1981) A gel electrophoresis method for quantifying the binding of
proteins to specific DNA regions: application
to components of the Escherichia coli lactose
operon regulatory system. Nucleic Acids Res 9
(13):3047–3060
4. Hellman LM, Fried MG (2007) Electrophoretic mobility shift assay (EMSA) for detecting
protein-nucleic acid interactions. Nat Protoc 2
(8):1849–1861. https://doi.org/10.1038/
nprot.2007.249
5. Berger R, Duncan MR, Berman B (1993)
Nonradioactive gel mobility shift assay using
chemiluminescent detection. BioTechniques
15(4):650–652
6. Hsieh YW, Alqadah A, Chuang CF (2016) An
optimized protocol for electrophoretic mobility shift assay using infrared fluorescent
dye-labeled oligonucleotides. J Vis Exp
117:54863. https://doi.org/10.3791/54863
7. Jing D, Agnew J, Patton WF et al (2003) A
sensitive two-color electrophoretic mobility
shift assay for detecting both nucleic acids and
protein in gels. Proteomics 3(7):1172–1180.
https://doi.org/10.1002/pmic.200300438
8. Rye HS, Drees BL, Nelson HC, Glazer AN
(1993) Stable fluorescent dye-DNA complexes
in high sensitivity detection of protein-DNA
interactions. Application to heat shock transcription factor. J Biol Chem 268
(33):25229–25238
338
James A. W. Stowell et al.