References
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assays for hit identification in drug discovery.
Anal Biochem 332(1):153–159. https://doi.
org/10.1016/j.ab.2004.04.031
2. Mashalidis EH, Sledz P, Lang S, Abell C
(2013) A three-stage biophysical screening cascade for fragment-based drug discovery. Nat
Protoc 8(11):2309–2324. https://doi.org/
10.1038/nprot.2013.130
3. Niesen FH, Berglund H, Vedadi M (2007) The
use of differential scanning fluorimetry to
detect ligand interactions that promote protein
stability. Nat Protoc 2(9):2212–2221. https://
doi.org/10.1038/nprot.2007.321
4. Martinez Molina D, Jafari R, Ignatushchenko
M et al (2013) Monitoring drug target engagement in cells and tissues using the cellular thermal shift assay. Science 341(6141):84–87.
https://doi.org/10.1126/science.1233606
5. Jafari R, Almqvist H, Axelsson H et al (2014)
The cellular thermal shift assay for evaluating
drug target interactions in cells. Nat Protoc 9
(9):2100–2122. https://doi.org/10.1038/
nprot.2014.138
6. Eglen RM, Reisine T, Roby P et al (2008) The
use of AlphaScreen technology in HTS: current
status. Curr Chem Genomics 1:2–10. https://
doi.org/10.2174/1875397300801010002
7. Almqvist H, Axelsson H, Jafari R et al (2016)
CETSA screening identifies known and novel
thymidylate synthase inhibitors and slow intracellular activation of 5-fluorouracil. Nat Commun 7:11040. https://doi.org/10.1038/
ncomms11040
8. Shaw J, Leveridge M, Norling C et al (2018)
Determining direct binders of the androgen
receptor using a high-throughput cellular thermal shift assay. Sci Rep 8(1):163. https://doi.
org/10.1038/s41598-017-18650-x
9. Shaw J, Dale I, Hemsley P et al (2019) Positioning high-throughput CETSA in early drug
discovery through screening against B-Raf and
PARP1. SLAS Discov 24(2):121–132. https://
doi.org/10.1177/2472555218813332
10. Alexandrov AI, Mileni M, Chien EY et al
(2008) Microscale fluorescent thermal stability
assay for membrane proteins. Structure 16
(3):351–359. https://doi.org/10.1016/j.str.
2008.02.004
11. Menzen T, Friess W (2013) High-throughput
melting-temperature analysis of a monoclonal
antibody by differential scanning fluorimetry in
the presence of surfactants. J Pharm Sci 102
(2):415–428. https://doi.org/10.1002/jps.
23405
12. Schulz MN, Landstrom J, Hubbard RE (2013)
MTSA--a Matlab program to fit thermal shift
data. Anal Biochem 433(1):43–47. https://
doi.org/10.1016/j.ab.2012.10.020
Quantitative Detection of Ligand Binding by DSF and CETSA HT
215
1. Lo MC, Aulabaugh A, Jin G et al (2004) Evaluation of fluorescence-based thermal shift
assays for hit identification in drug discovery.
Anal Biochem 332(1):153–159. https://doi.
org/10.1016/j.ab.2004.04.031
2. Mashalidis EH, Sledz P, Lang S, Abell C
(2013) A three-stage biophysical screening cascade for fragment-based drug discovery. Nat
Protoc 8(11):2309–2324. https://doi.org/
10.1038/nprot.2013.130
3. Niesen FH, Berglund H, Vedadi M (2007) The
use of differential scanning fluorimetry to
detect ligand interactions that promote protein
stability. Nat Protoc 2(9):2212–2221. https://
doi.org/10.1038/nprot.2007.321
4. Martinez Molina D, Jafari R, Ignatushchenko
M et al (2013) Monitoring drug target engagement in cells and tissues using the cellular thermal shift assay. Science 341(6141):84–87.
https://doi.org/10.1126/science.1233606
5. Jafari R, Almqvist H, Axelsson H et al (2014)
The cellular thermal shift assay for evaluating
drug target interactions in cells. Nat Protoc 9
(9):2100–2122. https://doi.org/10.1038/
nprot.2014.138
6. Eglen RM, Reisine T, Roby P et al (2008) The
use of AlphaScreen technology in HTS: current
status. Curr Chem Genomics 1:2–10. https://
doi.org/10.2174/1875397300801010002
7. Almqvist H, Axelsson H, Jafari R et al (2016)
CETSA screening identifies known and novel
thymidylate synthase inhibitors and slow intracellular activation of 5-fluorouracil. Nat Commun 7:11040. https://doi.org/10.1038/
ncomms11040
8. Shaw J, Leveridge M, Norling C et al (2018)
Determining direct binders of the androgen
receptor using a high-throughput cellular thermal shift assay. Sci Rep 8(1):163. https://doi.
org/10.1038/s41598-017-18650-x
9. Shaw J, Dale I, Hemsley P et al (2019) Positioning high-throughput CETSA in early drug
discovery through screening against B-Raf and
PARP1. SLAS Discov 24(2):121–132. https://
doi.org/10.1177/2472555218813332
10. Alexandrov AI, Mileni M, Chien EY et al
(2008) Microscale fluorescent thermal stability
assay for membrane proteins. Structure 16
(3):351–359. https://doi.org/10.1016/j.str.
2008.02.004
11. Menzen T, Friess W (2013) High-throughput
melting-temperature analysis of a monoclonal
antibody by differential scanning fluorimetry in
the presence of surfactants. J Pharm Sci 102
(2):415–428. https://doi.org/10.1002/jps.
23405
12. Schulz MN, Landstrom J, Hubbard RE (2013)
MTSA--a Matlab program to fit thermal shift
data. Anal Biochem 433(1):43–47. https://
doi.org/10.1016/j.ab.2012.10.020
Quantitative Detection of Ligand Binding by DSF and CETSA HT
215