THE NEWSLETTER OF ACTIVE MOTIF — OCTOBER 2003 • VOLUME 4 • NUMBER 4

Chromatin Immunoprecipitation using
General Transcription Factors
However, ChIP analyses can be technicallychallenging and difficult to validate unlessperformed using well-proven reagents. To
make validating your ChIP reagents easier,the new ChIP-IT Kit contains antibodies andcontrols that have been proven to work
effectively in ChIP. Plus, ChIP-IT antibodies
are directed against general transcription
factors so you get a more accurate analysis
of promoter activity than is possible withhistone-based ChIP kits. The new ChIP-IT Kit
is the only kit available today that contains
proven antibodies, reagents and controls,
so you can get the best results possible,
whatever your needs.
continued on page 5

Colorimetric Quantitation of MAPK-regulated
Transcription Factors
Active Motif’s new TransAM™ MAPK Family
Kit makes it possible to rapidly profile thelevels of six different transcription factorsthat are regulated by MAPK kinase activity.
In one simple experiment, you can assay
the levels of activation obtained under different growth and stimulation conditions.
Because TransAM is an ELISA-based colorimetric method*, it eliminates the use of radioactivity and provides quantitative resultsin less than five hours. This makes TransAM
faster, more sensitive and simpler thanother techniques for studying transcription
factors, like gelshift and Western blotting.
And, the TransAM method makes it possible
to study stimulated tissue samples.

Downstream MAPK detection
Activation of Mitogen Activated Protein
Kinase (MAPK) involves a three kinasecascade consisting of a MAP kinasekinase (MAPKKK or MEKK) that activatesa MAP/ERK kinase (MAPKK or MEK). This
stimulates a phosphorylation-dependent
increase in the activity of the MAP kinase.
Upon activation, MAP kinases phosphorylatea variety of intracellular targets includingtranscription factors. The TransAM MAPK
Family Kit directly assays the transcription
factors ATF-2, c-Jun, c-Myc, Elk-1, MEF2 and
STAT (Figure 1), all of which are regulated by
MAP kinase cascades and are key markers incell signaling.

1

Chromatin Immunoprecipitation using General Transcription
Factors1

Colorimetric Quantitation of
MAPK-regulated Transcription
Factors

Specificity for More
2 Higher
Effective Gene Silencing
Specific Detection of
3 Simple,
Epidermal Growth Factor
Receptor Phosphorylation
Measurement of Ku
6 Quantitativeand G/T Binding Protein
Delivers Affordable,
7 TimeLogic
Genome-scale BLAST Solutions
NO Production Faster8 Measureand More Accurately

2

Activation (OD450 nm)

Active Motif’s new ChIP-IT Kit for chromatin immunoprecipitation (ChIP) enables
researchers to get easy access to one ofproteomics’ powerful new tools for analyzing genome regulation. ChIP involves theidentification of DNA fragments that arebound by a particular transcription factor.
The fragments of interest are analyzed
by semi-quantitative PCR using fragmentspecific PCR primers or by hybridization togene arrays. The power of ChIP lies in the
combination of specificity (immunoprecipitation), sensitivity (PCR) and screening
power (array profiling). No other technique
can offer you such a versatile combination.

IN THIS ISSUE

Blank
Stimulated extract

1.5

1

0.5
0

c-Myc

MEF2

ATF-2

Elk-1

STAT

Figure 1: TransAM profiling MAPK-regulated transcription factors.
The TransAM MAPK Family Kit was used to assay the activation levelsof c-Myc, MEF2, ATF-2, Elk-1 and STAT using 5 µg/well nuclear extract
prepared from either HeLa (TPA treated), Jurkat (unstimulated) or
U-937 (TPA + IFNγ treated) cells.

continued on page 4

www.activemotif.com

Gene Silencing

Higher Specificity for More Effective Gene Silencing
Active Motif’s custom gripNA™ oligonucleotides bind to nucleic acids with higher
affinity and more stringent mismatch
discrimination than other gene silencing
reagents. This improves the results of yourgene silencing experiments by minimizing non-specific interactions that cancause unintended, mutant phenotypes.
gripNAs have proven to be effective atsilencing genes in zebrafish, mammaliancells, Xenopus and in in vitro translation
systems. Because gripNAs are resistant tonuclease degradation, they are stable in
the in vivo environment, enabling themto silence genes expressed over a range ofdevelopmental stages. And, you can deliver
gripNAs into cells using Chariot™ II, lipids,
electroporation, microinjection and thescrape method, making gripNAs the mostversatile tool available for gene silencing.
Enhanced PNA chemistry
gripNAs are a negatively charged form ofPeptide Nucleic Acids (PNAs). PNAs are
DNA analogs in which the nucleoside bases
are attached to a synthetic backbone
rather than to deoxyribose, as in DNA.
While PNAs bind complementary DNA andRNA by conventional Watson-Crick basepairing, they hybridize with greater affinity
and increased sequence specificity. Theirbinding affinity is significantly reduced (orcompletely eliminated) if there is evena single base-pair mismatch with theirtarget nucleic acid (Table 1). This minimizes
the non-specific silencing of genes withsequences that are nearly identical to yourtarget sequence.
Why choose gripNAs?
• Unsurpassed sequence specificity• Resistant to nucleases• Flexible synthesis modifications• Multiple delivery options• Simple online ordering

2

DNA/DNATm (°C)
ΔTm (°C)

gripNA/DNA
Tm (°C)
ΔTm (°C)

No mismatches

63.9

–

58.6

–

5´-CAC-TGA-CTT-GAG-ACC-A-3´

Mismatch A

57.0

6.9

41.3

17.3

5´-CAC-TGA-GTT-GAG-ACC-A-3´

Mismatch B

49.7

14.2

No Tm

–

5´-CAC-TGA-GTG-GAG-ACC-A-3´

Sequence of DNA oligonucleotide

Mismatch C

61.8

3.1

54.6

4.0

5´-CAC-TGA-CTT-GAG-ACG-A-3´

Mismatch D

56.7

7.2

No Tm

–

5´-CGG-TGA-CTT-GAG-ACC-A-3´

Mismatch E

53.6

10.3

No Tm

–

5´-CAC-TGA-CGT-GAG-ACC-A-3´

Mismatch F

56.2

7.1

No Tm

–

5´-CAC-TGA-CTG-GAG-ACC-A-3´

Mismatch G

54.2

9.7

42.4

16.2

5´-CAC-TGA-CAT-GAG-ACC-A-3´

Table 1: Increased binding specificity of gripNAs.
Identical 16-mer gripNA and DNA probes were synthesized with the sequence 5´-TGG-TCT-CAA-GTC-AGT-G-3´. These were annealed to a complementary DNA
oligo (5´-CAC-TGA-CTT-GAG-ACC-A-3´) in Hybridization Buffer (20 mM Tris, pH 7.5, 500 mM NaCl, 10 mM MgCl2). Each sample was heated to 90°C for 3 minutes,
then cooled gradually to room temperature. The samples were then heated at a rate of 1°C per minute from 20°C to 100°C using a thermal control unit linked to aspectrophotometer. Changes in A260 were recorded and a melting temperature (Tm) was calculated for the DNA/DNA and gripNA/DNA duplexes. The experiment
was then repeated by hybridizing the gripNA and DNA probes to a series of DNA oligos containing one- or two-base mismatches (shown above, with the mismatches underlined). Tm values were measured for each DNA/DNA and gripNA/DNA duplex and a ΔTm was calculated by subtracting the difference in the melting
temperatures of the complementary and mismatched probes. Samples that are unable to form a stable duplex generate a “No Tm” value in this assay and no ΔTm.

Hit your target only
A recurring problem of many gene silencing
reagents is a lack of specificity. This cancause unintended phenotypes because
these reagents may bind and silence
expression of more than just the intended
target. In vivo mismatch discrimination experiments have shown that gripNA binding
is reduced or eliminated by the presence ofa single base-pair mismatch. Recent experiments performed in zebrafish embryos
indicate that this specificity can improveyour results. While wild-type gripNA andmorpholino probes were comparable inpotency at silencing the chordin, uroDand no tail genes, the intentional inclusionof 2 and 4 base-pair mismatches in themorpholino probes caused non-specificeffects that were not caused by comparable gripNA probes.1

Order anytime
Active Motif’s custom gripNA synthesis
service can provide you with better materials for your gene silencing experiments. All
the information needed to design, orderand use gripNA probes can be accessed atwww.activemotif.com/gripna. Our simple,
online ordering system makes it easy toplace your gripNA order 24 hours a day, 7days a week. gripNA probes can be orderedwith or without Chariot II. In addition, you
can add a 3´ modification (Biotin, FITC or
primary amine) to your probe. Each gripNA
is verified by mass spectrophotometry andsupplied with a fluorescently labeled positive control for human CREB. Log on now
and order your custom gripNA today.
1. Urtishak et al. (2003) Developmental Dynamics 228(3).

Product

FormatCatalog No.

Custom gripNA™ Probe

200 nmol

24001

Custom gripNA™ Probe w/Chariot™ II

200 nmolPrimary Amine Modification

24002
24004

Biotin Modification

24005

Fluorescein Modification

24006

Chariot™ II

96 rxns

24008

gripNA™ Human CREB Positive Control

25 nmol

24007

gripNA™ Chordin Positive Control

5 nmol24009

Toll Free — 1 877 222 9543

OCTOBER 2003 • VOLUME 4 • NUMBER 4

Cell-based ELISAs

Simple, Specific Detection of Epidermal Growth Factor Receptor Phosphorylation

Regulation of epidermal growth factor receptor (EGFR) activity via phosphorylationof tyrosine 992 has been shown to be
critical for normal functioning of thiskey cellular receptor. Indeed, importantdisease states such as cancer have been
attributed to aberrant control of Y992
phosphorylation. This, and its cell-surface
location, has made EGFR a promising target
for anti-cancer therapeutics.
The FACE method
In the FACE method, cells are cultured in
96-well plates and stimulated to induce thepathway of interest. Following stimulation,
the cells are fixed, which preserves protein
modifications, including phosphorylation.
Each well is then incubated with a primary antibody specific for the activatedprotein of interest. Subsequent incubation with secondary HRP-conjugatedantibody and developing solution provides an easily quantified, colorimetric or
chemiluminescent readout. The signals arethen normalized for cell number using theprovided Crystal Violet solution and plotted. FACE Kits are easy to use and requireless than two hours hands on time, giving
you convenience not available with otherphosphorylation assays.
Specific antibodies
In order to get the most accurate results
possible, it is essential that antibodies
which bind only to the protein or protein
modification of interest be used in detection. This is even more important whenusing ELISA-based techniques that do not

involve the visualization of protein bands.
All antibodies included in FACE Kits are extensively tested and compared to Western
analysis to ensure your analysis is the mostaccurate and sensitive possible (Figure 1).
1

Figure 1: Western analysis of phosphorylated EGFR.
The phospho-EGFR antibody contained within FACE EGFR Kits wasused in Western analysis to verify antibody specificity. (Multiple bands
present in lanes 1 and 2 are due to EGF glycosylation).
Lane 1: 8 µg whole-cell extract from EGF-induced A-431 cells.
Lane 2: 8 µg whole-cell extract from non-induced A-431 cells.

Sensitivity without compromise
Achieving the sensitivity required to detectsome low-abundance kinases and kinaseregulated proteins can be challenging. In thepast, a lack of sensitivity has typically meantthat scientists must either load more ofeach sample, or change the cellular model.
Both of these solutions are far from optimal.
The improved sensitivity of FACE compared
to standard techniques, means that you

0.15

0.1

0.05
0

0

10
30
2
Time of Induction (minutes)

Figure 2: Detection of EGFR phosphorylation using FACE.
The FACE EGFR Kit was used to assay the level of phosphorylated
EGFR contained within EGF stimulated HeLa cells. FACE assays were
performed before and 2, 10 and 30 minutes after EGF induction.

Product

FormatCatalog No.

FACE™AKT

1 x 96 rxns
5 x 96 rxns
1 x 96 rxns
5 x 96 rxns

48120
48620
48220
48720

1 x 96 rxns
5 x 96 rxns
1 x 96 rxns
5 x 96 rxns

48150
48650
48250
48750

1 x 96 rxns
5 x 96 rxns
1 x 96 rxns
5 x 96 rxns

48140
48640
48240
48740

1 x 96 rxns
5 x 96 rxns
1 x 96 rxns
5 x 96 rxns

48110
48610
48210
48710

1 x 96 rxns
5 x 96 rxns
1 x 96 rxns
5 x 96 rxns

48100
48600
48200
48700

FACE™AKT Chemi
FACE™ EGFRFACE™ EGFR Chemi
FACE™ ERK1/2
FACE™ ERK1/2 Chemi
FACE™ JNKFACE™ JNK Chemi
FACE™ p38
FACE™ p38 Chemi

www.activemotif.com

2

won’t have to make such compromises
ever again. To demonstrate the sensitivity
achieved when using the colorimetric FACE
Kits, levels of phosphorylated EGFR present
in HeLa cells stimulated using EGF were
measured and compared to an equivalentWestern blot. Despite loading significant
amounts of sample (20 µg per well) andprolonged development, detection ofphosphorylated EGFR by Western blot
was not possible. In contrast, detection ofphosphorylated EGFR using FACE was simple
and quantitative (Figure 2). And, for those
who require even more sensitivity, all FACE
Kits are also available in an ultra-sensitive
chemiluminescent format. Don’t compromise, when you can use FACE Kits today.

Absorbance (OD450 nm)

Fast Activated Cell-based ELISA (FACE™)
Kits are an innovative alternative toclassical methods for monitoring protein
phosphorylation. FACE Kits enable modification-specific analysis directly within thecell without the need for time-consuming
cell extractions, gel electrophoresis or
membrane blotting. FACE Kits are designed
to overcome the limitations of classicalassays and at the same time improve thequality of your results.

3DNA-binding ELISAs

continued from page 1 — Colorimetric Quantitation of MAPK-regulated Transcription Factors
The TransAM method
TransAM MAPK Family Kits utilize a 96-wellplate, with each well containing six different double-stranded oligonucleotides.
When nuclear or whole-cell extract is added, the various MAPK-regulated transcription factors bind to the consensus-bindingsite on their particular oligonucleotide.
Primary antibodies specific for the activated forms of each transcription factor arethen added to individual wells, followed byincubation with secondary HRP-conjugatedantibody and developing reagent. The
plate is then read on a spectrophotometer,
which provides a quantitative, colorimetricreadout (Figure 2). Thus, TransAM MAPK
Family Kit makes it possible to rapidly
profile the levels of each MAPK-regulatedtranscription factor under various growth
and treatment conditions.
Improved method
TransAM assays are a marked improvement over other techniques used to study
transcription factor activation. Unlike
gelshift, Western blotting and reporter
gene methods, TransAM does not use
inefficient cloning and cell transfections,time-consuming gel exposures or radioactive probes. Inconsistencies due to variable
reporter plasmid transfection and theneed to construct stable cell lines are also
eliminated. TransAM assays are complete inmere hours, rather than in days, are moresensitive and provide quantitative results.
Moreover, TransAM can be used to study
stimulated tissue samples.
Product

Figure 2: Flowchart of the TransAM procedure.

TransAM advantages• Non-radioactive, colorimetric method
provides quantifiable results• Results in less than 5 hours• 10-fold greater sensitivity than gelshift• Simultaneous profiling of multiple
family members• Ability to assay cell and tissue samples

New TransAM Kits for c-Myc & Elk-1
In addition to the new TransAM MAPK
Family Kit, Active Motif is introducing two individual TransAM Kits for
c-Myc and Elk-1. Because these twotranscription factors are directly related
to MAPK cascades, they are widely
studied. The new TransAM Kits enable
users to study the activation of c-Myc
and Elk-1 individually.

The TransAM Family
In addition to the new TransAM MAPK Family Kit, Active Motif offers TransAM Family
Kits for simultaneously studying membersof the AP-1, GATA, HNF, NFκB, IRF and STAT
transcription factor families. TransAM Kits
for monitoring the individual transcription
factors NFκB** p50 and p65, HIF-1, c-Fos,
FosB, c-Jun, PPARγ, p53, MyoD, Sp1, Sp3,
C/EBP, NF-YA, ER, STAT3, c-Myc, Elk-1,*

FormatCatalog No.

TransAM™ MAPK Family

1 x 96-well plate

47296

TransAM™ c-Myc

1 x 96-well plate5 x 96-well plates

43396
43896

TransAM™ Elk-1

1 x 96-well plate5 x 96-well plates

44396
44896

4

NFATc1, and total and phosphorylated CREB
are also available. Ultra-sensitive TransAM
Chemiluminescent Kits are also available for
NFκB p50 and p65. For a more convenientand better method to study transcription
factor activation, try TransAM Kits.

Technology covered under AAT-filed patents and licensed exclusively
to Active Motif.
** Use of TransAM in NFκB-related drug discovery may be covered
under U.S. Patent No. 6,150,090 and require a license from Ariad
Pharmaceuticals (Cambridge, MA, USA).

Toll Free — 1 877 222 9543

OCTOBER 2003 • VOLUME 4 • NUMBER 4

Chromatin Immunoprecipitation

continued from page 1 — Chromatin Immunoprecipitation using General Transcription Factors
Transcriptional regulation is one of themain methods used by cells to control gene
expression. Traditionally researchers haveanalyzed transcription factors and genepromoter activity using methods such as reporter gene assays, EMSA, Western blotting
and more recently, using DNA microarrays.
However, although these methods have leadto significant advances in the scientific understanding of transcription, there is still aneed for improved assays. Specifically, there
is a need for better understanding of in vivointeractions between DNA and transcription
factors. The ChIP method overcomes thislimitation through a formaldehyde crosslinking step, which “freezes” and preserves
cellular protein/DNA interactions.
The complete solution
Classically, ChIP has been performed using
antibodies directed against abundant
chromatin components, such as acetylated
histones. However, while this method can
indicate the transcriptional activity of agiven promoter, it does not reveal the
specific transcription factor bound to thepromoter(s) of interest. In contrast, ChIP
performed using transcription factor-specific antibodies enables the direct monitoring of transcription factor/DNA interactions.
However, this method is technically morechallenging and requires the preparationof several complicated buffers, inhibitors
and blocking reagents. Preparation of these
individual components within the lab can bea time consuming and expensive undertaking. Unlike other commercially available ChIP
kits, Active Motif’s ChIP-IT Kit provides you
with all the reagents and controls needed
to successfully perform your ChIP experiments. Plus, the ChIP-IT Kit also contains
positive control antibody directed against
the general transcription factor, transcription factor IIB (TFIIB). Because TFIIB binds
to nearly all active promoters, it is an ideal
positive control if you are trying to validate
other transcription factor-specific antibodies. No other kit available today can provide
you with such a complete solution.

www.activemotif.com

Cross-link protein to DNA inliving cells with formaldehyde

Break open cells
and shear DNA

Add pre-blocked
Protein G Agarose Beads

Reverse cross-links

Immunoprecipitate to enrich
for fragments bound byprotein of interest

Add primary
antibody of interest

Detect and quantify
precipitated DNA through
PCR and hybridization methods

Figure 1: Schematic of Chromatin Immunoprecipitation.

The ChIP method
In the ChIP-IT method, intact cells are fixedusing formaldehyde, which cross-links andtherefore preserves protein/DNA interactions. DNA is then sonicated into small
uniform fragments and the DNA/protein
complexes are immunoprecipitated using anantibody directed against the DNA-binding
protein of interest. Following immunoprecipitation, cross-linking is reversed and the
DNA is screened to determine which genes,or groups of genes, were bound by theprotein of interest (Figure 1). The versatilityof ChIP means that screening can be doneusing generalized hybridization or a moretargeted PCR-based approach, dependingon the needs of your experiment.
Proven effective
Successful ChIP depends on sonicated DNAof the correct length and antibody(ies)that are effective at immunoprecipitating
protein/DNA complexes. However, it canbe difficult to determine whether “pulldown” was successful or not, unless you
possess an antibody that has already been
proven effective in ChIP. So that time spenton antibody validation is minimized, the
TFIIB antibody included in the ChIP-IT Kit
has been rigorously tested and proven to“pull down” TFIIB-bound DNA. And, because TFIIB binds almost all active promoters, you can be certain that if your DNA issuitable for ChIP, it will work with the ChIPIT Kit. Using the ChIP-IT Kit there will be noneed to waste time and money wondering
whether your ChIP has worked or not.
Controls for confidence
Specificity of antibody binding is essential
for getting the most accurate data fromyour ChIP experiments. It is important thatany non-specific DNA precipitation, which
can result in incorrect analyses, is easily
identified. As such, the ChIP-IT Kit contains
several PCR primers, which are directedagainst DNA known to either bind TFIIB
or not. And, because the TFIIB antibody is
known to be highly specific in ChIP you cananalyze your DNA to ensure “pull-down”
was specific. Collectively, these proven reagents will help ensure the validity of yourresults. To get the most out of your ChIP
experiments, buy the ChIP-IT Kit today.

Product

Catalog No.

ChIP-IT Kit

53001

5

DNA Repair

Quantitative Measurement of Ku and G/T Binding Protein
Active Motif’s new DNA Repair Kits utilize
a novel method* that makes the study of
DNA repair proteins faster and more sensitive. Unlike conventional techniques suchas EMSA and Western blot, these kits are
DNA-binding ELISAs that provide quantitative results in less than five hours, whileeliminating the use of radioactivity. Studying DNA repair has never been so simple.
Ku and G/T binding protein (GTBP) aredamage recognition proteins that bind asheterodimers to DNA with specific lesions.
Once bound, these proteins catalyze repairof the DNA and have been shown to be
critical in maintaining genome integrity.
Despite the interest in studying DNA damage and repair, there is a lack of convenient, sensitive assays that are suitable formodern high-throughput research. That’s
why we have developed the new Ku70/86
and GTBP DNA Repair Kits.
Advantages• Non-radioactive, colorimetric method• 10X more sensitive than EMSA
• Results in under 5 hours• Works with cells and tissues• High-throughput compatible
GTBP

Absorbance (OD450 nm)

A

1.25

1

Simple, efficient method
The DNA Repair Kit method is simple andefficient and can be used with both tissue
and cell culture extracts. Each kit includes
a 96-well plate in which multiple copies ofa specific double-stranded oligonucleotide
have been immobilized. The oligonucleotide in the GTBP Kit contains a G/T mismatch, while that in the Ku70/86 Kit simply
has a blunt end. When cellular extract is
added, the repair protein of interest binds
to the oligonucleotide. Each well is thenincubated with a primary antibody thatis specific for the repair protein being
studied. Addition of a secondary HRP-conjugated antibody and developing solution
provides an easily quantified colorimetricreadout (Figure 1). Now you can efficiently
monitor the regulation of DNA repair
proteins under different treatments.
Easily quantitate your data
Being able to measure small changes in
DNA repair protein activity is important.
While traditional methods such as Western

0.75

0.5

blot are sensitive, they typically provide
qualitative, rather than quantitative data.
Active Motif’s DNA Repair Kits, however,provide a colorimetric readout that is
easily quantified, so you can detect eventhe slightest change in protein activity. To
illustrate, GTBP activity was measured andcompared using Active Motif’s GTBP DNA
Repair Kit and by Western blot. The GTBPDNA Repair Kit results are clearly morequantitative than Western blot (Figure 2).
Simplify your detection
Active Motif’s new Ku and GTBP DNA
Repair Kits will simplify the way you study
interactions between damaged DNA andrepair proteins. These easy-to-use kits
are flexible enough to use in a variety offunctional studies, such as examining thespecificity of repair proteins for a particular
DNA damage. For a better method to study
DNA repair protein activity, try one of
Active Motif’s new DNA Repair Kits.
*

Patent Pending.

0.25
0

Raji

PANC-1

JurkatB

Figure 2: Measurement of GTBP activity in different cell lines using
Western blot and the new GTBP DNA Repair Kit.
Raji, PANC-1 and Jurkat nuclear extracts were assessed for GTBP activity using Western blot (A) and the new GTBP DNA Repair Kit (B).

6

Figure 1: Flowchart of the DNA Repair Kit procedure.

Product

FormatCatalog No.

GTBP DNA Repair Kit

1 x 96-well plate5 x 96-well plates

51096
51596

Ku70/86 DNA Repair Kit

1 x 96-well plate5 x 96-well plates

51196
51696

Toll Free — 1 877 222 9543

OCTOBER 2003 • VOLUME 4 • NUMBER 4

BLAST Enhancement

TimeLogic® Delivers Affordable, Genome-scale BLAST Solutions

TimeLogic’s acceleration technology makes
it possible to process extremely large-scale
genomic analyses with BLASTN or BLASTP,
and also employ rigorous, frame-translated
methods for more effective searching ofdivergent sequences (T-BLASTX).

The BLAST system is driven by DeCypher®,an innovative combination of software andreconfigurable hardware that processes
trillions of base comparisons per second.
Beyond dramatic BLAST analysis speed, thesystem offers many advantages:• Greater default sensitivity than NCBIBLAST: Find more hits per search• Smaller footprint: The computing
capability of a server farm in the spaceof a single computer• Low ownership costs: Unlike clusters,
DeCypher brings no increased costs for
power, cooling and maintenance

The TimeLogic BLAST System is affordable
for departmental budgets, and installs rapidly within a server from Sun Microsystems
or Dell Computers. Please contact us to findout how the TimeLogic BLAST System canaccelerate your research goals.
Contact Information
TimeLogic Corporation
U.S. +1 775 833-0200 (PDT)
http://www.timelogic.com

Visit our web site to find out about additional TimeLogic BLAST, HMM and SmithWaterman Systems.

BLAST Comparison of SNP Data with the Human Genome
The aim of pharmacogenomics is to develop
drugs that can be specifically targeted to apatient’s genotype group for high efficacy
response and no side effects. Extensive
progress in mapping SNPs, which are single
nucleotide polymorphisms occurring inover 1% of a population, has brought rapid
advances to this nascent field.
Identifying SNPs involved in novel diseasetargets, whether in a coding (cSNP) or regulatory (rSNP) capacity, is a key focus. With
the help of population-based SNP studies,
pharmacogenomic therapies and potential
preventative approaches can be designed toaddress genetic risk factors.
Comparing a large collection of SNP data tomonthly Human genome updates can drain
an institution’s computational resources.
However, this is a critical task for researchgroups pursuing pharmacogenomics-based
therapies.
To illustrate the BLAST System’s speed andefficiency for pharmacogenomics, population genetics and modern genetic toxicology, TimeLogic recently performed a private,large-scale benchmark. A BLASTN search wasused to compare 1.7 million 25-mer nucleic
sequences of proprietary SNP data (a total

30
28 days

25

Days

TimeLogic was recently acquired by Active
Motif to help researchers connect wet
lab science and computational biology.
TimeLogic’s BLAST System accelerates
algorithms critical for genomic analysis,
SNP identification and all-to-all database
comparisons. It can bring, within a single
server, the performance of hundreds ofCPUs. With an intuitive web interface andcommand line access for batch searching,
BLAST is a powerful tool for all levels of
bioinformatics expertise.

20

15
10
5
28 hours0

BLAST
SystemLinux Cluster(30 x PIII CPUs)

Figure 1: BLAST System vs. a Linux Cluster.
17 million 25-mers compared to the human genome with BLASTN.

of 42,500,000 bases) against the humangenome target of 3,099,043,584 bases.
The DeCypher BLAST Solution completed
the search in 28 hours, or 24X faster than aLinux cluster of 30 Pentium III 1 GHz CPUs
(Figure 1). This test demonstrates performance equivalent to a server farm of over
700 CPUs. For additional details on thisbenchmark and other performance measures, please visit:
http://www.timelogic.com/performance/.

• Simple scalability: Increase performance as your search needs growsimply by adding additional DeCypher
acceleration cards

www.activemotif.com

7

Nitric Oxide Quantitation

Measure NO Production Faster and More Accurately
the nitrite into a purple-colored azo
compound that is quantitated using aspectrophotometer. However, excess
NADPH, an essential cofactor in nitrate
reduction, interferes with the Griess
reaction, limiting the sensitivity of thetwo-step assay. To improve sensitivity, an
LDH step can be added before the Griess
Reagent step. Though this increases thesensitivity of the assay, it is more timeconsuming and complicated.

Advantages• Increased sensitivity• Fast and easy to use• Wider dynamic range• High-throughput compatible• Works with a variety of sample types

A faster procedure
Our Nitric Oxide Quantitation Kit offersa better alternative than conventional
Two- and Three-Step NO assays. The kit
contains a unique formulation of cofactorsthat accelerate the conversion of nitrate
to nitrite, while simultaneously degrading
NADPH. This decreases the reductase
step to just 30 minutes and eliminates thecomplicated, time-consuming third step.

The old way has more steps
Typically, NO assays are performed using
either a two-step assay or a three-steplactate dehydrogenase (LDH) assay. In bothmethods, the first step is the reductionof nitrate into nitrite by nitrate reductase.
In the final step, Griess Reagent converts

8

Get yours today
Active Motif’s Nitric Oxide Quantitation
Kit makes NO detection quick andeasy. Simplify the way you measure NO
production and order a Nitric Oxide
Quantitation Kit today!
1.2
1.0
OD540 nmActive Motif’s Nitric Oxide Quantitation
Kit provides a fast and sensitive method formeasuring the production of nitric oxide(NO) in your samples. The kit employs
an innovative cofactor technology that
reduces the time and number of steps
needed to measure NO levels. Plus, theNitric Oxide Quantitation Kit has a wider
dynamic range of NO measurement thanconventional technology (Figure 1), giving
you more accurate results.

Active Motif's NO Kit
LDH method

0.8
0.6
0.4
0.2
0.0

0 5 10 15 20 25 30 35 40 45 50 55 60

Concentration of Nitrate (µM)
Figure 1: Dynamic range of nitrate standard curves.
Nitrate standard curves produced using the Nitric Oxide Quantitation
Kit and a conventional Three-Step LDH-method kit.

Product

FormatCatalog No.

Nitric Oxide Quantitation Kit

2 x 96 rxns

40020

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