support the solubility of the ligand through a large concentration range. In the case of a small-molecule ligand, solubilizing
agents (e.g., DMSO) are sometimes required, and we recommend that the concentrations of such agents be kept low (e.g.,
<5% DMSO) so that the protein receptor is minimally affected
by them. Importantly, as formulated in this chapter, any additives included in this buffer but not in the protein buffer will be
halved when the protein is added. Thus, if the receptor is
thought to tolerate 5% DMSO, 10% could be included in this
buffer.
2. We ordinarily prefer highly purified proteins for MST, i.e.,
>95% pure as judged by a sodium dodecyl sulfate polyacrylamide gel stained with Coomassie Blue. Indeed, with the proteins used in this protocol, our usual practice is to purify them
using gel-filtration chromatography. However, we have noticed
no significant difference in the results using the unpurified
proteins, as described in this protocol.
3. Although spectrophotometers are available with very short
path lengths and very small volumes, we do not recommend
their use for this protocol. Experience has demonstrated that
the best accuracy is attained by using a cuvette-based
spectrophotometer.
4. The extinction coefficients were calculated from the aminoacid sequences of the respective proteins using the method of
Pace and coworkers [12]. This method is encoded in the
webserver ProtParam (www.expasy.org/protparam). It
requires knowledge of the amino-acid sequence of the protein.
5. We recommend taking a full spectrum of the protein solutions
from 250 to 400 nm. The reading at 333 nm should be
doubled and subtracted from that at 280 nm to arrive at the
A 280 corrected for scattering of aggregates in solution [12].
6. Many other dyes are available; we have had success with Alexa
488 as well. In addition to NHS esters, maleimide versions of
dyes may be used to label cysteine residues. Also, there are dyes
that are able to bind specifically to histidine tags. The instrument’s manufacturer, NanoTemper Technologies, sells a number of dyes specially designed for use in MST.
7. Both pieces of software are adequate for this protocol. Most
MST instruments in the field have MO.Control, but we prefer
NT.Control because of its increased flexibility for advanced
users. We present instructions for both pieces of software in
this chapter.
8. This dilution step may not be needed. Because of the high
efficiency with which α-CT can be labeled, it is necessary to
dilute the dye to prevent disruption of the SBTI/α-CT
178
Shih-Chia Tso and Chad A. Brautigam
agents (e.g., DMSO) are sometimes required, and we recommend that the concentrations of such agents be kept low (e.g.,
<5% DMSO) so that the protein receptor is minimally affected
by them. Importantly, as formulated in this chapter, any additives included in this buffer but not in the protein buffer will be
halved when the protein is added. Thus, if the receptor is
thought to tolerate 5% DMSO, 10% could be included in this
buffer.
2. We ordinarily prefer highly purified proteins for MST, i.e.,
>95% pure as judged by a sodium dodecyl sulfate polyacrylamide gel stained with Coomassie Blue. Indeed, with the proteins used in this protocol, our usual practice is to purify them
using gel-filtration chromatography. However, we have noticed
no significant difference in the results using the unpurified
proteins, as described in this protocol.
3. Although spectrophotometers are available with very short
path lengths and very small volumes, we do not recommend
their use for this protocol. Experience has demonstrated that
the best accuracy is attained by using a cuvette-based
spectrophotometer.
4. The extinction coefficients were calculated from the aminoacid sequences of the respective proteins using the method of
Pace and coworkers [12]. This method is encoded in the
webserver ProtParam (www.expasy.org/protparam). It
requires knowledge of the amino-acid sequence of the protein.
5. We recommend taking a full spectrum of the protein solutions
from 250 to 400 nm. The reading at 333 nm should be
doubled and subtracted from that at 280 nm to arrive at the
A 280 corrected for scattering of aggregates in solution [12].
6. Many other dyes are available; we have had success with Alexa
488 as well. In addition to NHS esters, maleimide versions of
dyes may be used to label cysteine residues. Also, there are dyes
that are able to bind specifically to histidine tags. The instrument’s manufacturer, NanoTemper Technologies, sells a number of dyes specially designed for use in MST.
7. Both pieces of software are adequate for this protocol. Most
MST instruments in the field have MO.Control, but we prefer
NT.Control because of its increased flexibility for advanced
users. We present instructions for both pieces of software in
this chapter.
8. This dilution step may not be needed. Because of the high
efficiency with which α-CT can be labeled, it is necessary to
dilute the dye to prevent disruption of the SBTI/α-CT
178
Shih-Chia Tso and Chad A. Brautigam