Use of Activity-Based Probes to Develop High
Throughput Screening Assays That Can Be Performed in
Complex Cell Extracts
Edgar Deu1, Zhimou Yang1, Flora Wang2, Michael Klemba2, Matthew Bogyo1,3*
1Department of Pathology, Stanford School of Medicine, Stanford, California, United States of America, 2Department of Biochemistry, Virginia Polytechnic Institute and
State University, Blacksburg, Virginia, United States of America, 3Department of Microbiology and Immunology, Stanford School of Medicine, Stanford, California, United
States of America
Background: High throughput screening (HTS) is one of the primary tools used to identify novel enzyme inhibitors.
However, its applicability is generally restricted to targets that can either be expressed recombinantly or purified in large
Methodology and Principal Findings: Here, we described a method to use activity-based probes (ABPs) to identify
substrates that are sufficiently selective to allow HTS in complex biological samples. Because ABPs label their target
enzymes through the formation of a permanent covalent bond, we can correlate labeling of target enzymes in a complex
mixture with inhibition of turnover of a substrate in that same mixture. Thus, substrate specificity can be determined and
substrates with sufficiently high selectivity for HTS can be identified. In this study, we demonstrate this method by using an
ABP for dipeptidyl aminopeptidases to identify (Pro-Arg)2-Rhodamine as a specific substrate for DPAP1 in Plasmodium
falciparum lysates and Cathepsin C in rat liver extracts. We then used this substrate to develop highly sensitive HTS assays
(Z’.0.8) that are suitable for use in screening large collections of small molecules (i.e .300,000) for inhibitors of these
proteases. Finally, we demonstrate that it is possible to use broad-spectrum ABPs to identify target-specific substrates.
Conclusions: We believe that this approach will have value for many enzymatic systems where access to large amounts of
active enzyme is problematic.
Citation: Deu E, Yang Z, Wang F, Klemba M, Bogyo M (2010) Use of Activity-Based Probes to Develop High Throughput Screening Assays That Can Be Performed
in Complex Cell Extracts. PLoS ONE 5(8): e11985. doi:10.1371/journal.pone.0011985
Editor: Floyd Romesberg, The Scripps Research Institute, United States of America
Received June 13, 2010; Accepted July 15, 2010; Published August 5, 2010
Copyright: ? 2010 Deu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by funding by NIH grants R01 EB005011 and R01 AI078947 and a New Investigator Award in Pathogenesis from the Burrough
Wellcome Fund (all to MB). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
One of the most common techniques used by the pharmaceu-
tical industry to identify novel drug leads is high throughput
screening (HTS). This method allows inhibition effects of large
numbers of compounds to be determined in a relative short period
of time. HTS assays have traditionally been performed with either
a recombinant form of the target enzyme or with purified native
enzyme . More recently, HTS has been performed using both
cell-based and extract-based assays [2,3]. While these types of
assays avoid the need to express and purify a target enzyme, they
often rely on genetically engineered reporter systems that tend to
have a high rate of false positives. To get around this problem, it is
possible to enhance the expression level of the targeted activity to
reduce the background noise of the system . Regardless, a
specific inhibitor (often identified using recombinantly expressed
enzyme) [1,5] or a genetic knock-out of the target enzyme [4,6] is
needed to prove that the assay is target-specific. Therefore, in
almost all cases, these assays have been developed for targets or
systems that are amenable to genetic manipulation and/or protein
However, not all organisms are genetically tractable, and many
enzymes cannot be purified or produced recombinantly in an
active form. This is especially true for enzymes that are naturally
expressed as zymogens and require posttranslational modification
(proteolytic cleavage, phosphorylation, glycosylation, etc.) to
become active, or those for which specific interactions with
cellular components are required (protein-protein interaction,
cofactors, etc.). Activity-based probes (ABPs) are ideally suited to
assess binding and inhibition of target enzymes in the context of
complex protein mixtures. Because they covalently modify the
catalytic residue of the targeted enzyme, they can be used in
competition assays to assess both potency and selectivity of
compounds in intact cells, extracts and even whole organisms
[7,8]. However, the readout for such assays requires SDS-PAGE
to measure residual target labeling by the probe. Therefore, this
approach is not suitable for use in HTS. Alternatively, a recent
study demonstrated the use of ABPs as reporters of enzyme activity
for HTS. This study demonstrated that measuring changes in
fluorescence anisotropy of the tag on an ABP as it binds its target
can provide a sufficiently sensitive and quantitative readout of
labeling to allow HTS . Because labeling of the target is used as
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the readout of the assay, it is particularly valuable for enzymes for
which suitable substrates have not been identified. However, this
approach requires expressed or purified enzymes because the
background of probe labeling in crude extracts is often high.
Alternatively, once a sufficiently selective substrate can be
identified for a desired target enzyme, it is possible to directly
measure its inhibition in complex mixtures. Here we demonstrate
the use of ABPs to assess the selectivity of reporter substrates in
crude cell extracts. We demonstrate that this approach facilitates
the identification of substrates whose kinetics of turnover inhibition
perfectly correlate with the kinetics of labeling of the target
enzyme by the ABP. Such substrates can be deemed to be selective
for the target enzyme and can therefore be use for HTS. In this
study, we demonstrate the application of this method using an
ABP that targets dipeptidyl aminopeptidases. Specifically, we use
the probe in crude cell extracts from the human malaria parasite
Plasmodium falciparum and in crude rat liver extracts to identify a
highly selective substrate for dipeptidyl aminopeptidase 1 (DPAP1)
and cathepsin C (Cat C). We then demonstrate that this substrate
can be used to develop a highly sensitive and stable assay that is
suitable for use in HTS with large libraries of small molecules.
We were initially interested in developing an assay that could be
used in HTS to identify inhibitors of DPAP1, a protease that is
expressed by the human malaria parasite Plasmodium falciparum.
This enzyme is an essential cysteine protease involved in the final
stages of hemoglobin degradation [10,11]. DPAP1 is refractory to
genetic disruption  and its inhibition blocks parasite growth
both in vitro and in vivo , thus making it a potentially valuable
anti-malaria drug target. We also chose to focus on this target
because proteases are generally difficult to express in their active
form as most of them are translated as inactive pro-enzymes.
Furthermore, expression of P. falciparum proteins is especially
challenging due to the A/T-rich nature of the genome of this
organism. Additionally, DPAP1 is an ideal enzyme to demonstrate
the value of our approach because we already identified a suitable
ABP for this target [12,13], we had specific information about its
substrate specificity [10,12,14], and we had access to recombi-
nantly expressed enzyme for comparison of our method with
standard screening assays .
DPAPs recognize the N-terminal amine of substrate proteins
and are efficient at cleaving dipeptide fluorogenic substrates. Our
SAR studies on DPAP1 indicated that proline at P2 (N-terminal
amino acid) provides selectivity towards DPAP1 relative to other
cysteine proteases in P. falciparum such as DPAP3 or the falcipains
[10,12]. Additionally, the screen of a positional scanning library of
7-amino-4-methylcoumarin (AMC) substrates identified Arg as a
preferred residue at P1 . Therefore, we synthesized the (Pro-
Arg)2-Rho substrate to target DPAP1 in parasite lysates
(Figure 1A). This substrate is cleaved twice by DPAP1 to produce
We chose a rhodamine-based substrate because free rhoda-
mine emits at a wavelength (523 nm) that is high enough to be
free from most of the auto-fluorescence background of com-
pounds in a diverse library of small molecules. Using more
traditional protease fluorogenic substrates, such as AMC-
substrates for example, which emit at a lower wavelength, will
likely result in an increase in the rate of false negatives during
HTS, since a significant portion of the molecules in a library will
likely emit light below 500 nm. Also, a bidentate substrate is likely
to be more specific since it needs to be cleaved twice in order to
produce an optimal signal.
To begin to assess the utility of this substrate for measuring
DPAP1 activity in complex protein mixtures, we measured its
apparent Kmvalue in parasite extracts (Figure 1B). Importantly,
we obtained an apparent Km value (36 mM) that was within
experimental error of the value measured using recombinant
DPAP1, suggesting that this substrate functions similarly against
the native and recombinant enzymes. To test whether (Pro-Arg)2-
Rho is processed specifically by DPAP1, we treated parasite lysates
for 1 h with increasing concentrations of FY01 - a fluorogenic
ABP developed in our lab to target dipeptidyl aminopeptidases
[12,13] - and measured both DPAP1 labeling (Figure 1C) and
substrate turnover (Figure 1D). Because FY01 only labels DPAP1
in trophozoite lysates (Figure 1C) it was straightforward to quantify
labeling of the enzyme by SDS-PAGE. We found that the dose
dependent labeling of DPAP1 by FY01 perfectly correlates with
the extent of inhibition of (Pro-Arg)2-Rho turnover (Figure 1D)
suggesting that the substrate is processed virtually exclusively by
Having demonstrated the selectivity of our substrate, we next
wanted to determine if it could be used in a low-volume, 384-well
plate assay. For the assay we continuously monitored substrate
turnover for 5 minutes and then used the slope of the emission
fluorescence over time as readout of enzyme activity. As a positive
inhibition control, we used JCP410, a covalent inhibitor previously
identified in our lab to be highly selective for DPAP1 . Using
this inhibitor and the continuous measurement of substrate
processing, we could demonstrate that the assay has a signal-to-
noise ratio (S/N) of 300 and an almost perfect Z’ factor (Figure 2A).
Because end-point assays are generally preferred when screening
large numbers of compounds, we adapted the assay into an end-
point format by simply quenching the reaction after 10 min with
acetic acid (data not shown). We evaluated this end-point assay in
a fully automated format using a multi-channel peristaltic pump to
dispense reagents into a 384-well plate, and a stackable plate
reader. The end-point assay has a slightly lower S/N than the
continuous assay, but the Z’-factor remains very high (0.9)
(Figure 2B). Therefore, both the continuous and endpoint assays
using the (Pro-Arg)2-Rho substrate are highly sensitive assays that
are suitable for use in HTS with a large numbers of compounds.
Furthermore, the use the 384 well format allows screening of large
libraries using a relatively small amount of parasite lysates.
To demonstrate the general utility of our approach, we also
applied the same methods to rat liver extracts. These samples are
more complex than parasite lysates and contain a number of
highly related papain fold cysteine proteases, in addition to the
DPAP1 homolog Cat C. As expected, addition of FY01 to rat liver
lysates resulted in labeling of multiple proteases targets (Figure 3A).
However, only the labeling of rat Cat C (at 23 kDa) correlated
with inhibition of (Pro-Arg)2-Rho turnover, suggesting that it is a
highly selective substrate for Cat C in this system (Figure 3B).
Furthermore, when the rat liver assay was converted to a 384-well
plate format (20 mL reaction volume), we were also able to
generate a high S/N and Z’ factor making it suitable for use in
HTS (Figure 3C). Overall, these results demonstrate that our
identified substrate can be used in multiple different extract
systems and furthermore that ABPs that target multiple related
enzymes can be used to identify specific substrates.
HTS remains one of the most powerful methods for identi-
fication of small molecule inhibitor leads. Before embarking on a
large-scale screen, it is important to develop an assay that is
sufficiently robust to allow the generation of meaningful results.
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For most enzymatic targets, HTS assays make use of highly
purified proteins and well-defined substrates. This requirement
for pure enzyme prevents the use of HTS for a large number of
difficult to express proteins. For enzymes such as proteases,
expression of mature, active enzymes is often challenging. This
can be further compounded for organisms such as P. falciparum
whose proteins typically do not express well in heterologous
expression systems due to its high A/T-rich genome. Therefore,
developing methods that allow rapid development of sensitive
and robust assays that can be performed in crude protein extracts
would enhance the number of targets that are accessible to HTS.
In this study, we demonstrate that ABPs can be used to identify
enzyme substrates that are sufficiently selective for use in HTS
While the results presented here focus on protease targets, it
should also be possible to use any of the ever-increasing number of
ABPs (for review see [8,15,16]) to develop similar assay for other
classes of enzymes. We demonstrate here that ABPs need not be
selective for the target of interest but can bind many related
targets. Since it is possible to correlate substrate processing with
specific labeled proteins using an SDS-PAGE readout, the only
limitation is the ability to resolve the probe-labeled proteins.
Another possible limitation is access to a set of suitable substrates
for a target of interest. However, because ABPs generally bind to
Figure 1. Use of an ABP to identify a DPAP1-selective substrate in parasite lysates. A. Structure and reaction mechanism of the (Pro-Arg)2-
Rho substrate. B. Measurement of (Pro-Arg)2-Rho apparent Kmin trophozoite lysates (circles) and with recombinant DPAP1 (triangle). Turnover rates
at increasing concentrations of substrate were fitted to a Michaelis-Menten equation as described in the methods section. C. Labeling of DPAP1
activity in parasite lysates with FY01. Trophozoite lysates were incubated for 1 h with increasing concentrations of FY01. Labeling was stopped by
boiling the sample in SDS-PAGE loading buffer. DPAP1 activity was measured using a flatbed fluorescent scanner. D. DPAP1 labeling correlates with
substrate turnover inhibition. An aliquot of the samples treated for 1 h with FY01 was diluted in assay buffer containing 10 mM of (Pro-Arg)2-Rho, and
the initial turnover rate was measured in a 96-well plate (circles). This turnover rate is plotted with the labeling quantified in C.
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target enzyme using similar mechanism as native substrates, they
often serve as a starting points for design of substrate scaffolds. In
addition, in the course of designing selective probes, a substantial
amount of structure/activity information is usually generated.
Thus, probes can be used to design substrates and vice versa. Even
in situations where a perfectly selective substrate cannot be easily
identified, it may be possible to use inhibitors to reduce the activity
of other unwanted enzymes that contribute to substrate processing.
Our approach is likely to be most successful for enzymatic
activities that are highly abundant in an extract, either because the
targeted enzyme is highly expressed or because it is the most
efficient catalyst of substrate turnover. We anticipate that large
and structurally diverse libraries of substrates can be used to
identify specific substrates for enzymes that have low abundance
or reduced catalytic efficiency.
to any biological sample that can be obtained in sufficient quantity. A
number of ABPs that target specific classes of enzymes have already
been developed and can be used for this approach immediately. We
believe that this approach willbe useful to identify novel inhibitors for
a variety of drug targets that cannot be expressed recombinantly, as
well as to decrease the cost associated with HTS.
Materials and Methods
Synthesis of (Pro-Arg)2-Rho
The synthesis was based on previously described syntheses of
similar rhodamine-based substrates [17,18]. Rhodamine110 was
reacted with Fmoc-Arg(Pbf)OH (6 eq) and pyridine (6 eq) for 24 h to
yield [Fmoc-Arg(Pbf)]2-Rho. After purification by flash chromatog-
raphy (1% methanol in dichloromethane), the 9-fluorenylmethylcar-
bamate (Fmoc) group was deprotected in a 1:1 mixture of
acetonitrile:diethylamine. HPLC purified [HN-Arg(Pbf)]2-Rho was
the presence of 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium
(HBTU; 6 eq), 1-hydroxybenzotriazole (HOBT; 6 eq), and
Diisopropylethylamine (DIEA;18 eq) to yield [Boc-Pro-Arg(Pbf)]2-
Rho. After deprotection of the t-butyloxycarbamate (BOC) in
Frifluoracetic acid (TFA):H2O: tetrahydrofuran (THF) (95:2.5:2.5),
(Pro-Arg)2-Rho was precipitated in cold ether and purified by high
pressure liquid chromatography (HPLC). The substrate purity was
determined by liquid chromatography/mass spectrometry (LC/MS)
to be .95% pure.
Cell lysates preparation
D10 P. falciparum parasites were cultured synchronously as
described in . Parasites were harvested at trophozoite stage
(,34 h post RBC invasion) by lysing the red blood cell (RBC)
membrane with 0.15% saponin in phosphate buffered saline (PBS)
and spinning down the parasite pellet. Those were lysed in 2
volumes of 1% nonidet P40 in PBS for 1 h in ice. The soluble
fraction of the lysates was store at 280uC. Rat liver extracts were
obtained as described in .
Labeling of cysteine protease activity with ABPs
Extracts from P. falciparum or rat livers (purchased from
Rockland Immunochemicals, Gilbertsville, PA) were diluted 10-
fold in assay buffer (50 mM sodium acetate pH 5.5, 5 mM MgCl2,
and 5 mM dithiothreitol (DTT)) and treated for 1 h with FY01 at
room temperature. No APLAC approval was required to obtain
rat liver extracts. Samples were boiled in SDS-loading buffer and
run in an SDS-PAGE gel. Labeled proteins were detected in a
9410 Typhoon flatbed scanner (Ammersham Bioscience, GE
Healthcare). The identity of the different bands in the gels for
parasite and liver lysates has been described in  and ,
Continous fluorogenic assays
All assay were performed in assay buffer with 10 mM of (Pro-
Arg)2-Rho substrate in 96- or 384-well plates (100 or 20 mL
reaction volume, respectively), depending on whether the assay
was used to validate the substrate as a target-specific substrate, or
to test the assay for HTS purposes, respectively. The reaction
was run with 1% of parasite lysates or with 0.1% of rat liver
extract. Substrate turnover was measured for 5 min at 523 nm
( lexcitation=492 nm) in a Spectramax M5 plate-reader (Molecular
Devices). Recombinant DPAP1 was prepared as described in .
End-point assay for DPAP1
This assay was performed in 384-well plates. The compound
addition, reagent dispensing and fluorescent readout of this end-
Figure 2. Development of a DPAP1-specific HTS assay. A. Continuous assay. The assay was carried out in 384-well plates using 1% of parasite
lysates. Substrate turnover was continuously measured for 5 min. JCP410 (10 mM) was used as a positive inhibition control. Z’ factor, S/N, and % CV of
the negative control are shown. B. End-point assay for HTS. The reaction described in A was quenched after 10 min by addition of 0.5 M acetic acid.
The final concentration of rhodamine product was quantified by fluorescence.
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Figure 3. Cat C-specific fluorogenic assay in rat liver lysates. A. Labeling of Cat C with FY01. Rat liver extract extracts were treated with
increasing concentrations of FY01 for 1 h and labeled proteins analyzed by SDS-PAGE followed by scanning of the gel using a flatbed laser scanner.
The location of labeled Cat C is indicated. B. Inhibition of substrate turnover specifically correlates with Cat C labeling. The cleavage of (Pro-Arg)2-Rho
substrate was measured prior to analysis of FY01 labeling shown in part A. Quantification of the indicated labeled proteins relative to DMSO control is
shown. C. Cat C-specific HTS assay in rat liver extracts. Rat liver lysates were treated for 30 min with either DMSO or JCP410 (10 mM) followed by the
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point assay was fully automated. The reagents were added using a Download full-text
stackable dispenser equipped with a multichannel peristaltic
pump (Matrix WellMate Bulk Dispenser). 10 mL of 2%
trophozoite lysates in assay buffer were added to 10 mL of
20 mM (Pro-Arg)2-Rho in assay buffer. The plate was spin for
10 s to ensure that all the reaction volume was at the bottom of
the well. After 10 min, the reaction was stopped by adding 20 mL
of 0.5 M acetic acid. Fluorescence was measured at 530 nm
(lexcitation=485 nm) with an Analyst AD stackable plate-reader
Statistic evaluation of the HTS assays
As a positive inhibition control, we used 10 mM of JCP410,
which is a known covalent inhibitor of DPAP1  and Cat C
. The inhibitor was added to parasite lysates at the same time
as the substrate, or to rat liver extracts 30 min prior to the
addition of the substrate. The Z’ factor and coefficient of
variation (% CV) values were evaluated according to eqs 1 and 2,
where the positive and negative superindexes refer to the positive
(JCP410) and negative (DMSO) inhibition controls (SD =
The authors thank Dr. Kenny Ang, Dr. Steven Chen and Dr. Michelle
Arkins at the Small Molecule and Discovery Center (UCSF) for their help
in the implementation of the DPAP1 HTS.
Conceived and designed the experiments: ED MB. Performed the
experiments: ED. Analyzed the data: ED MB. Contributed reagents/
materials/analysis tools: ZY FW MK. Wrote the paper: ED MB.
1. Williams KP, Scott JE (2009) Enzyme assay design for high-throughput
screening. Methods Mol Biol 565: 107–126.
2. Zock JM (2009) Applications of high content screening in life science research.
Comb Chem High Throughput Screen 12: 870–876.
3. An WF, Tolliday N (2010) Cell-based assays for high-throughput screening. Mol
Biotechnol 45: 180–186.
4. Rosse G, Kueng E, Page MG, Schauer-Vukasinovic V, Giller T, et al. (2000)
Rapid identification of substrates for novel proteases using a combinatorial
peptide library. J Comb Chem 2: 461–466.
5. Luciani N, de Rocquigny H, Turcaud S, Romieu A, Roques BP (2001) Highly
sensitive and selective fluorescence assays for rapid screening of endothelin-
converting enzyme inhibitors. Biochem J 356: 813–819.
6. Wakata A, Lee HM, Rommel P, Toutchkine A, Schmidt M, et al. (2010)
Simultaneous fluorescent monitoring of proteasomal subunit catalysis. J Am
Chem Soc 132: 1578–1582.
7. Berger AB, Vitorino PM, Bogyo M (2004) Activity-based protein profiling:
applications to biomarker discovery, in vivo imaging and drug discovery.
Am J Pharmacogenomics 4: 371–381.
8. Fonovic M, Bogyo M (2007) Activity based probes for proteases: applications to
biomarker discovery, molecular imaging and drug screening. Curr Pharm Des
9. Bachovchin DA, Brown SJ, Rosen H, Cravatt BF (2009) Identification of
selective inhibitors of uncharacterized enzymes by high-throughput screening
with fluorescent activity-based probes. Nat Biotechnol 27: 387–394.
10. Deu E, Leyva MJ, Albrow V, Rice MJ, Ellman JA, et al. (2010) Functional
studies of the Plasmodium falciparum dipeptidyl aminopeptidase I (DPAP1)
using small molecule inhibitors and active site probes. Chemistry and Biology In
11. Klemba M, Gluzman I, Goldberg DE (2004) A Plasmodium falciparum
dipeptidyl aminopeptidase I participates in vacuolar hemoglobin degradation.
J Biol Chem 279: 43000–43007.
12. Arastu-Kapur S, Ponder EL, Fonovic UP, Yeoh S, Yuan F, et al. (2008)
Identification of proteases that regulate erythrocyte rupture by the malaria
parasite Plasmodium falciparum. Nat Chem Biol 4: 203–213.
13. Yuan F, Verhelst SH, Blum G, Coussens LM, Bogyo M (2006) A selective
activity-based probe for the papain family cysteine protease dipeptidyl peptidase
I/cathepsin C. J Am Chem Soc 128: 5616–5617.
14. Wang F KP, Deu E, Bibb B, Lauritzen C, Pedersen J, Bogyo M, Klemba M
(2010) Definition of the catalytic role of dipeptidyl aminopeptidase 1 in
hemoglobin catabolism in the Plasmodium falciparum food vacuole. Molecular
and Biochemical Parasitology, In Submission.
15. Evans MJ, Cravatt BF (2006) Mechanism-based profiling of enzyme families.
Chem Rev 106: 3279–3301.
16. Cravatt BF, Wright AT, Kozarich JW (2008) Activity-based protein profiling:
from enzyme chemistry to proteomic chemistry. Annu Rev Biochem 77:
17. Leytus SP, Melhado LL, Mangel WF (1983) Rhodamine-based compounds as
fluorogenic substrates for serine proteinases. Biochem J 209: 299–307.
18. Liu J, Bhalgat M, Zhang C, Diwu Z, Hoyland B, et al. (1999) Fluorescent
molecular probes V: a sensitive caspase-3 substrate for fluorometric assays.
Bioorg Med Chem Lett 9: 3231–3236.
19. Greenbaum D, Medzihradszky KF, Burlingame A, Bogyo M (2000) Epoxide
electrophiles as activity-dependent cysteine protease profiling and discovery
tools. Chem Biol 7: 569–581.
addition of 10 mM of (Pro-Arg)2-Rho. The turnover rate was continuously measured for 5 min in a 384-well plate. Z’ factor, S/N, and % CV of the
negative control are shown.
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