THE NEWSLETTER OF ACTIVE MOTIF — MAY 2003 • VOLUME 4 • NUMBER 2

Simple, Efficient Analysis of ERK1/2 Phosphorylation
Fast Activated Cell-based ELISA (FACE™) Kits*
provide a simple, sensitive and efficientmethod to monitor protein phosphorylation.
FACE Kits enable you to perform modification-specific analysis directly within the cell,making them the simplest phospho-specificassay available. This also means there’s noneed for time-consuming cell extractions,gel electrophoresis or membrane blotting,which saves you both time and money.
The extracellular signal-regulated kinases
1 and 2 (ERK1 and ERK2) are importantmembers of the mitogen activated protein

kinase (MAPK) family of proteins that arefound in all eukaryotes. The ERK1/2 signalingcascade has been shown to be a criticalregulator of cell differentiation, cell physiology and neural function. Loss of regulatory
control of these proteins has been implicated in a variety of pathological conditions, including cancer and autoimmune
diseases. Yet, despite widespread interest in
ERK1/2 function, there is a lack of sensitive, convenient assays that are suitable formodern high-throughput drug discovery
research. That’s why the scientists at Active
Motif developed the FACE ERK1/2 Kit.
continued on page 4

IN THIS ISSUE
1

Simple, Efficient Analysis of
ERK1/2 Phosphorylation

1

Colorimetric Quantitation of
Activated IRF Family Members

Accurate Monitoring of2 Rapid,IκBα Phosphorylation
More Effective Gene
3 Get
Silencing with gripNAs™
cient Protein Transfection
6 Effiusing Chariot™
More Accurate Measure7 Faster,ment of NO Production
Specific 6xHis Protein
8 MorePurification using Silica Resin

Active Motif’s new TransAM™ IRF Family Kit
makes it possible to rapidly profile the levels of four different IRF transcription factor
family members in one simple experiment,
and to compare the levels obtained under
different growth and stimulation conditions. Because TransAM is an ELISA-based,colorimetric method*, it eliminates the useof radioactivity and provides quantitative
results in less than five hours. This makes
TransAM faster, more sensitive and simpler
than other techniques, like gelshift andWestern blotting.
Interdependent familyInterferon Regulatory Factors (IRFs) are afamily of transcription factors that playa critical role in host defense. The manymembers of the IRF family are involved in
the regulation of interferon α and β, which
in turn regulate host immunity, cell growthand hematopoietic development. As thefunction and activity of individual IRFs candepend both on their activation state aswell as that of other IRF family members,
the ability of the TransAM IRF Family Kit tosimultaneously profile the activation statesof IRF-1, -2, -3 and -7 (Figure 1) is critical tounderstanding their role in the regulation of
immune function and oncogenesis.
The TransAM method
The TransAM method facilitates the studyof transcription factor activation in mammalian tissue and cell culture extracts. The
TransAM IRF Family Kit utilizes a 96-well

IRF activation (OD450 nm)

Colorimetric Quantitation of Activated IRF Family Members
2

Blank
U-937 + TPA + IFNγ

1.5
1
0.5
0

IRF-1

IRF-2 IRF-3 IRF-7
IRF family members

Figure 1: Profiling activation of various IRF family members.
The TransAM IRF Family Kit was used to assay IRF-1, -2, -3 and -7 activation using 5 µg/well nuclear extract prepared from U-937 cells that
had been cultured in medium supplemented with 5 nM TPA (phorbol,
12-myristate, 13 acetate) for 72 hours and stimulated with 5 ng/ml
interferon gamma for two hours at 37°C prior to harvesting.

continued on page 5

www.activemotif.com

Sandwich ELISAs

Rapid, Accurate Monitoring of IκBα Phosphorylation
Active Motif’s FunctionELISA™ IκBα Kit
provides a fast, accurate method to monitor the phosphorylation state of IκBα.
FunctionELISA Kits utilize a Sandwich ELISA
technique that is faster and simpler to perform than other techniques, like Western
blots. In just hours, the FunctionELISA IκBα
Kit enables sensitive, quantitative measurement of phosphorylated IκBα.
IκBα is widely studied because it is an
inhibitor of NFκB. In the majority of cells,
NFκB exists in an inactive form in thecytoplasm, bound to the inhibitory IκB
proteins. External stimuli, such as tumor
necrosis factor (TNF) or other cytokines,
initiate a signal transduction cascade that
leads, ultimately, to the degradation ofIκBα. The NFκB dimers then translocate
to the nucleus and activate target genes.
Thus, the FunctionELISA IκBα Kit provides
an accurate means both to monitor thephosphorylation state of IκBα and to correlate this information with the activationand translocation of NFκB.
HRP

E
Substrate

Secondary
Antibody

Detecting
AntibodyAg

CaptureAntibody

0

A

5
10
30
time (minutes)

60

chemiluminescence

200000B

160000120000
80000
40000
0

0

10

20

30
40
time (minutes)

50

60

70

Figure 2: Induction of IκBα phosphorylation with TNF-α.
Jurkat cells were grown to 2 x 106 cells/ml and treated with 1 nM TNF-α. Cells were harvested at various indicated time points and cell lysates were tested inWestern analysis (Fig. 2A) using an Phosphorylated-IκBα antibody (Active Motif Cat. No. 40904) and the FunctionELISA IκBα Kit (Fig. 2B).

Fast, convenient format
FunctionELISA IκBα offers a simple, rapid
method to monitor the phosphorylation
state of IκBα. The kit utilizes the Sandwich
ELISA technique, which utilizes two antibodies directed against different epitopes
to capture and quantifiably measurephosphorylated IκBα (Figure 1). Comparedto Western blotting, FunctionELISA is fasterand simpler to perform, as it does not require the running, blotting and developingof gels. Plus, the kit’s 96-well format makes
it possible to process multiple samples
quickly, at whatever level of throughput isbest for you.
The FunctionELISA advantage• Analyze multiple samples in low-volume, high-throughput experiments• Analysis complete in just hours• Provides more quantitative results

Capture Assay“Sandwich”

Quantitative results
The Capture Antibody in the FunctionELISAIκBα Kit specifically binds the phosphorylated form of IκBα. A polyclonal Detecting
Antibody is then used with an HRP-conjugated Secondary Antibody to measure
the bound IκBα. For increased sensitivity,
FunctionELISA IκBα Kits utilize a highly
sensitive chemiluminescent detection
method that uses a luminometer. As demonstrated, FunctionELISA provides resultsthat are much more quantitative thanthose that can be generated by Western
blot analysis (Figure 2).
Complete kit ensures reproducibility
FunctionELISA IκBα contains all of thereagents required to quickly quantify
phosphorylated IκBα in your sample,
including recombinant phospho-IκBαthat can be used to generate a standard
curve. For fast, accurate quantification of phosphorylated IκBα, try the
FunctionELISA IκBα Kit.

Figure 1: Sandwich ELISA schematic.

Product2

FormatCatalog No.

FunctionELISA™ IκBα

1 x 96-well plates
5 x 96-well plates

48005
48505

FunctionELISA™ TRAIL

1 x 96-well plates
5 x 96-well plates

48010
48510

FunctionELISA™ Cytochrome c

1 x 96-well plates
5 x 96-well plates

48006
48506

Toll Free — 1 877 222 9543

MAY 2003 • VOLUME 4 • NUMBER 2

Gene Silencing

Get More Effective Gene Silencing with gripNAs™
Active Motif’s custom gripNA™* oligonucleotides provide you with a better alternative for gene silencing. Traditional gene
inhibition reagents such as DNA-antisense,
siRNA, S-oligos and morpholinos often
suffer from mistargeting due to non-specific interactions as well as from difficulties
with transfection. However, gripNAs havesuperior sequence specificity, are resistantto nuclease degradation and offer simple
delivery with the use of Active Motif’s
novel protein delivery reagent, Chariot™ II.
This makes gripNAs the tool of choice foryour gene silencing experiments.

Proven results
gripNAs are proven effective in mammalian cells, Zebrafish, Xenopus and in in
vitro translation systems. To illustrate, asynthesized gripNA probe that was targetedagainst human cyclin B1 was delivered into
human fibroblast (HS-68) cells using Chariot
II. Western blot analysis performed 20 hours
post-delivery clearly indicates that thecyclin B1 signal has been inhibited (Figure 1).

Why gripNAs?
• Unsurpassed sequence specificity• Resistant to nucleases• Flexible synthesis modifications• Highly soluble• Easy delivery with Chariot II• Simple online orderingA

A better backbone
gripNA oligonucleotides are a novel formof negatively charged Peptide Nucleic
Acids (PNAs). PNAs are DNA analogs inwhich the nucleoside bases are attached
to a synthetic backbone rather than todeoxyribose, as in DNA. PNAs bind complementary DNA and RNA by Watson-Crickbase pairing with even higher affinities
than nucleic acids. PNAs can also invade
and bind double-stranded DNA and areresistant to nucleases. However, poor watersolubility has limited their effectiveness ingene silencing.
Improved synthesis
To overcome the limitations of traditional
PNAs, Effimov et al, developed PNAs withnegative charges1. The result is gripNAs,
which are highly soluble and bind RNA withhigh affinity and sequence specificity. This
reduces mistargeting and improves yourgene inhibition studies.

12

within your target cells. Active Motif’s
Chariot II delivery reagent offers simple,
efficient delivery of gripNAs with minimal
cytotoxicity (Figure 2), providing you with acomplete solution for gene silencing.

3

B
Figure 1: gripNA silencing of cyclin B1.
An 18-mer gripNA probe (1 µM) targeted against cyclin B1 was complexedwith Chariot II. gripNA probe alone and the Chariot II/gripNA probe
complex were overlaid onto cultured HS-68 cells synchronized by serum
starvation for 40 hr, then released by addition of serum for 4 hr. Expression of cyclin B1 protein was analyzed by Western blot after 20 hr (A).
Cdk2 protein was also analyzed by Western blot to normalize theamount of protein loaded in each lane (B). Data provided by Dr. L
Chalion, Dr. M. Morris and Dr. G. Divita, CNRS, Montpellier, France.
Lane 1:
Negative control
Lane 2:
Addition of gripNA probe only
Lane 3:
Addition of Chariot II/gripNA probe complex

Simple, non-cytotoxic delivery
The delivery of gene silencing reagents
into the cell is a major limitation for mostexperiments. Classical delivery mechanisms
such as microinjection and DNA/RNAtransfection can be time consuming andinefficient. These conventional methods
may also result in high levels of cytotoxicity

Figure 2: Chariot™ II delivery of Fluorescein-tagged gripNA.
An 18-mer gripNA probe (1 µM ) labeled on its 3´ end was complexedwith Chariot II in PBS and incubated for 30 min at 37°C, then overlaid
onto cultured HS-68 for 1 hour. Cells were washed extensively prior
to observation.

Order 24/7
All the information you need to design,order and use your gripNA probe can befound at www.activemotif.com/gripna.
gripNAs can be ordered 24 hours a day, 7days a week using our simple online ordering system. 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 to ensure your success. Log on
now and order your custom gripNA today.
1. Effimov et al. (1998) NAR 26: 566-575.

Product

FormatCatalog No.

Custom gripNA™ Probe

200 nmol

24001

Custom gripNA™ Probe w/Chariot™ II

200 nmol

24002

Primary Amine Modification

24004

Biotin Modification

24005

Fluorescein Modificationwww.activemotif.com

24006

gripNA™ Human CREB Positive Control

25 nmol

24007

Chariot™ II

96 rxns

24008

3

Cell-Based ELISAs

continued from page 1 — Simple, Efficient Analysis of ERK1/2 Phosphorylation

The FACE advantage• Simple, quantitative method• Fixing cells preserves the protein’s
activation state• Cell-based assay eliminates extractions
and gel electrophoresis• Increased target epitope exposure

ERK1/2 that is phosphorylated on Thr202
and Tyr204 or Thr185 and Tyr187 for ERK1
and ERK2, respectively. Using FACE Kits
you’ll achieve increased sensitivity andresults you can be confident in.
1.4
1.2
OD450 nm

The FACE™ method
FACE is a sensitive assay designed to detect
activated proteins within mammalian cells.
In the FACE method (Figure 1), cells arecultured in 96-well plates and stimulated toinduce the pathway of interest. Following
stimulation, the cells are fixed, washed andquenched. Each well is then incubated witha primary antibody specific for the activated protein of interest. Addition of secondary HRP-conjugated antibody and developing solution provides an easily quantified,colorimetric readout. The number of cells
in each well can be normalized simply withthe provided Crystal Violet solution. FACE
Kits are easy to use and require less than
2 hours of hands on time, giving you thefreedom to accelerate your research.

1

Phospho-ERK

0.8

Total-ERK

0.6
0.4
0.2
0

0

1000
100
10
PMA induction (ng/ml)

Figure 2: Measurement of phosphorylated and total ERK.
Murine Macrophage 4/4 cells were cultured in 96-well plates andserum-starved for 16 hours. Cells were then stimulated with the indicated amounts of Phorbol 12-myristate 13-acetate (PMA) for 10 minutes
and fixed. Total and phospho ERK were each assayed in triplicate using
the phospho and total ERK antibodies included in the FACE ERK1/2 Kit.
Data was plotted after correction for cell number (performed throughuse of Crystal Violet). Note that the induction treatment did notaffect the overall level of total ERK.

Instant fixation for accurate results
Preparation of cellular extracts prior toWestern blotting or ELISA analysis takes
at least one hour to perform. In addition,
Western analysis requires the determination of protein concentration prior to loading your gel. During this time, additional
protein modifications can occur, which willalter your results. FACE Kits, however, use
a fixation step that “freezes” the cellularstate. This means that there is no risk
of further protein modification before
analysis, and gives you a clear view of theprotein state within the cell at that specifictime point. Improve the accuracy of yourresults by using FACE.
FACE Kits are also available for monitoring
the cellular levels of phosphorylated p38,
JNK and AKT, with many other kits to be released throughout 2003. For a better way tomonitor protein phosphorylation, try FACE.
*

Developed in agreement with Dr. M. Peppelenbosch and Dr. H. Versteeg.

Specificity for confident conclusions
The excellent specificity and sensitivity
achieved using the FACE ERK Kit is demonstrated in Figure 2. To ensure that you
are able to achieve comparable results,
we test all antibodies included in FACE
Kits for cross-reactivity. The included
total-ERK1/2 antibody will recognize all
ERK1/2 present within the cell, regardlessof its phosphorylation state, whereas thephosphospecific antibody detects only
Figure 1: Flowchart of the FACE procedure.

4

Product

FormatCatalog No.

FACE™ ERK1/2

1 x 96 rxns
5 x 96 rxns

48140
48640

FACE™AKT

1 x 96 rxns
5 x 96 rxns

48120
48620

FACE™ JNK

1 x 96 rxns
5 x 96 rxns

48110
48610

FACE™ p38

1 x 96 rxns
5 x 96 rxns

48100
48600

Toll Free — 1 877 222 9543

MAY 2003 • VOLUME 4 • NUMBER 2

Transcription Factor ELISAs

continued from page 1 — Colorimetric Quantitation of Activated IRF Family Members
plate containing double-stranded oligonucleotide that includes the consensus-bindingsite for IRF. When nuclear or whole-cell
extract is added, the various members of the
IRF family bind to the oligonucleotide. Primary antibodies that are specific for the activated forms of each IRF family member arethen added to individual wells, followed byincubation with secondary HRP-conjugatedantibody and developing reagent. Reading
the plate on a spectrophotometer provides aquantitative, colorimetric readout (Figure 2).
Thus, the TransAM IRF Family Kit makes it
possible to rapidly profile the levels of eachfamily member under various growth andstimulation conditions (Figure 1, Page 1).
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
in mere hours, rather than days, are moresensitive and provide quantitative results.

Moreover, TransAM can be used to study
stimulated tissue samples.
The TransAM advantage• 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 cells or tissue samples
A family of families
In addition to the new TransAM IRF Family
Kit, Active Motif offers TransAM Family Kits
for simultaneously studying members of theAP-1, NFκB and STAT transcription factor
families. TransAM Kits for monitoring theindividual transcription factors NFκB** p50
and p65, HIF-1, c-Fos, FosB, c-Jun, PPARγ, p53,
MyoD, Sp1, Sp3, C/EBP, NF-YA, ER, STAT3,
NFATc1 and total and phosphorylated CREB
are also available. TransAM Chemi Kits thatare 100-fold more sensitive than gelshift
assays are available for NFκB p50 and p65.
For a 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).

Figure 2: Flowchart of the TransAM procedure.

Product
TransAM™ IRF Family

www.activemotif.com

FormatCatalog No.

2 x 96-well plates

45296

BIOBASE - The TRANSFAC® Company
BIOBASE is the leading content provider for gene regulation. Its industry
standard databases, TRANSFAC® andTRANSPATH®, provide expert knowledge for data analysis and utilization.
With long-standing experience in gene
regulation, BIOBASE offers researchers not only database subscriptions,but also scientific consulting. Over 100
years of scientific excellence in thisdomain are accumulated at BIOBASE tosolve your problems, royalty-free.
TRANSFAC – Transcription factor database
With over 25,000 entries, BIOBASE’s
TRANSFAC is the Gold Standard oftranscription factor databases. Curated
by biologists, TRANSFAC is comprisedof data from approximately 10,000
publications. Divided into two main
sections, TRANSFAC provides knowledge about the proteins and theirbinding sites. With an easy-to-use,
web-based user interface, TRANSFAC
offers encyclopedic knowledge in acompressed form, as well as tools forin-depth analysis of sequences andgene expression data.
TRANSPLORER™– Binding site elucidation
The Java application TRANSPLORER™
enables complete analysis of regulatory
DNA sequences. The integrated Dragon
Promoter Finder and Dragon Gene Start
Finder (from the Laboratories for Information Technology, Singapore) are used
to predict promoter regions in genomic
sequences. TRANSPLORER’s powerfulmatrix prediction engine allows immediate promoter analysis. Tightly linked
with TRANSFAC, TRANSPLORER is acomprehensive toolbox that enables
the scientist to analyze and understanda gene’s regulation.
For more information, visit BIOBASE’s
website at http://www.biobase.de orcontact their marketing department by
email at marketing@biobase.de or byphone at +49 (5331) 85 84 30.

5Protein Transfection

Efficient Protein Transfection using Chariot™
Chariot™ is a revolutionary protein delivery
reagent that efficiently transports biologically active proteins, peptides and antibodies directly into cultured mammalian cells in
less than two hours. The cells can be assayed
immediately after delivery to determine theeffects of the introduced material, without
the need for fixing. This makes Chariot ideal
for functional studies in living cells.

? How does Chariot work?
Chariot forms a non-covalent complex whencombined with a purified protein, peptide
or antibody. This complex stabilizes themacromolecule, protecting it from degradation during internalization. It also eliminates
the need for fusion proteins or chemical
coupling. After delivery, the complex dissociates in a process called “decaging”, leaving
the macromolecule biologically active andfree to proceed to its cellular target. The
delivery process is serum independent, giving you the flexibility to culture your cells inwhichever media type you prefer.

?

Does Chariot work with primarycells or even plant cells?

Chariot delivers effectively into primarycells, something that most other delivery
systems cannot do. Most notable is theuse of Chariot on hard to transfect primaryneurons (Jurney W. et al. Journal of Neuroscience 22: 6019-6028. 2002) without any
signs of cytotoxicity or rejection. Chariot
has also been shown to deliver proteins
into plant protoplasts (Wu, Yan, et al. The
Plant Journal. 33: 131-137. 2002).

?

What about protein functionality?

The ability of Chariot to deliver biologically active protein is shown using a 119 kDa
subunit of β-galactosidase. β-galactosidase
is composed of four subunits that must assemble to form the functional protein. HeLa
cells turn blue upon addition of X-gal following delivery of the Chariot-galactosidase
complex, demonstrating successful deliveryof functional β-galactosidase (Figure 1).

Log on to our website at www.activemotif.com
to download the protocol as well as the mostcurrent list of publications that cite Chariot.
there any size limit to the? Isproteinsthat Chariot can deliver?

Figure 1: Chariot delivery of β-galactosidase.
One µg of a 119 kDa subunit of β-galactosidase was complexed with
Chariot™ Protein Delivery Reagent (see page 6) for 30 minutes anddelivered into HeLa cells. Cells were fixed and stained with X-gal
2 hours post-delivery.

There is one limitation; peptides below 12
amino acids are too small for Chariot to efficiently deliver. However, we have not founda maximum size limitation. The key to remember is that the larger the protein or antibody,the longer it takes to get in; but it will get in.

are researchers using? WhatChariot for?

Chariot delivery use the? Doesendosomal pathway?

We have an ever growing list of citations
from researchers who have used Chariot intheir published research. Chariot is becoming the number one choice for protein,
peptide and antibody delivery into bothprimary and cultured cells. Peluso et altransfected a peptide and an antibody into
granulosa cells to study the PKCd-Dependent Pathway (Endocrinology. Vol. 142(10):4203-4211. 2001).

No, Chariot delivery is independent of theendosomal pathway. Temperature studies
have been performed at 37°C and at 4°C withalmost identical results. This demonstrates
that Chariot is independent of the endosomal
pathway as it is shut down at 4°C.

Product

FormatCatalog No.

Chariot™

25 rxns
100 rxns

30025
30100

6

Another recent publication shows theusage of Chariot in chick ganglions tostudy the coordinated activation of
autophosphorylation sites in RET Receptor Tyrosine Kinase (Coupier et al, JBC. Vol.
277, No. 3. 2002. 1991-1999). And Ikari et
al used Chariot to study up-regulation of
Sodium-dependent glucose transporter byinteraction with Heat Shock protein 70 inepithelial cells (JBC. Vol. 277, No. 5. 3333833343. 2002).

is the difference between? WhatChariot and Chariot II?
Chariot is intended for peptide, protein
and antibody delivery. New this year is
Chariot II, which is for gripNA delivery.
Chariot II has modifications that are optimized for use in transfecting gripNAs forgene silencing. To learn more about genesilencing with gripNAs, turn to page 3 inthis newsletter.

Toll Free — 1 877 222 9543

MAY 2003 • VOLUME 4 • NUMBER 2

Nitric Oxide Quantitation

Faster, More Accurate Measurement of NO Production
The Nitric Oxide Quantitation Kit is afaster, more sensitive method for measuring 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 tomeasure NO levels. Plus, the Nitric Oxide
Quantitation Kit has a wider dynamic rangeof NO measurement than conventional
technology, giving you more accurate results. And, the kit can be used with a large
variety of sample types, including plasma,serum, saliva, urine, cell lysate, tissue
homogenate and tissue culture medium.

spectrophotometry (Figure 1). However,assay sensitivity is limited when using the
Two-Step method because excess NADPH,
which is an essential cofactor in step one,interferes with the Griess Reagent in step
two. Assay sensitivity can be increased
through the addition of a third step, whereby lactate dehydrogenase (LDH) eliminates
excess NADPH prior to the Griess reaction.
However, while inclusion of a third LDH
step increases sensitivity, it is not optimal
as it also increases the time required toperform each assay.

Improved accuracy for better results
In addition to its increased speed and user
friendliness, the Nitric Oxide Quantitation
Kit provides more accurate measurement
over a wider range of sample concentration than possible with other currently
used methods. The improved linearity overa broader dynamic range (Figure 2) meansthat your results will be more precise withless optimization and repetition than ispossible with any other kit.
1.2
1.0

The old way
The best index of total NO production is
the sum of both the nitrite and nitrate,commonly quantified in a Two-Step assay.
The first step is the conversion of nitrate
to nitrite by the use of NADH or NADPHdependent nitrate reductase. Nitrite levels
are measured by the addition of Griess
Reagent, which converts nitrite into apurple-colored azo compound. NO concentration is then indirectly measured usingwww.activemotif.com

Nitric Oxide

Nitrate Reductase
NitriteAssay Using
Griess reagent
Figure 1: Measurment of NO by Griess Reagent.
Nitric oxide is converted to nitrite, then assayed using Griess Reagent.

Save time and money
The Nitric Oxide Quantitation Kit offers afaster and easier-to-use alternative to conventional Two- and Three-Step NO assays.
Active Motif’s Nitric Oxide Quantitation Kit
contains a unique formulation of cofactorsthat accelerate the conversion of nitrate
to nitrite, while simultaneously degrading
NADPH. The result is that the time required
to perform the reductase step is decreased
to only 30 minutes and there’s no need for
a complicated, time-consuming third step,which saves you both time and effort.

OD540 nmNitrateNO is a key molecule that, either directlyor through intracellular signaling, stimulates
host defenses in the immune system, maintains blood pressure in the cardiovascularsystem and modulates neural transmission
in the brain. Production of NO by Nitric
Oxide Synthase (NOS) can be regulated byphysiological changes in intracellular calcium
concentrations or induced in a cell-specificmanner. Monitoring of NO production incells is a useful tool for determining NOS activity. However, the extremely short half-lifeof NOS makes it unsuitable for detection.

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 2: Dynamic range of nitrate standard curves.
Nitrate standard curves produced using the Nitric Oxide Quantitation
Kit and a conventional Three-Step LDH-method kit.

Get better results today
The Nitric Oxide Quantitation Kit makes
NO measurement more sensitive, fasterand easier to perform than other, moreconventional methods. In addition, its
96-well format enables straightforward
high-throughput automation. For a bettermethod of measuring NO production, try
the Nitric Oxide Quantitation Kit today!

Product

FormatCatalog No.

Nitric Oxide Quantitation Kit

2 x 96 rxns

40020

7

6xHis-Tagged Protein Purification

More Specific 6xHis Protein Purification using Silica Resin
Active Motif’s Ni-TED Protein Purification
System is a versatile and powerful toolfor isolating recombinant 6xHis-tagged
proteins. Unlike traditional resins madeof agarose, Ni-TED uses a silica resin thatmakes purification of 6xHis proteins better
than ever before.
Increased specificity
Conventional protein purification using
agarose-based nickel columns is often characterized by co-purification of untagged
proteins due to non-specific binding to the
agarose matrix. Ni-TED’s silica resin has fewer side chains than agarose, which increases
the selectivity of Ni-TED and reduces thenon-specific binding of cellular proteins.
This results in a much cleaner purificationof your target protein (Figure 1).
Quick and easy
Ni-TED uses a simple one-step protocolthat is ideal for purifying 6xHis-tagged proteins produced in any expression system.
You can choose to elute your proteins under both native and denaturing conditions
by using either competition with imidazole
or a simple reduction in pH.

8

1

2

3

4

5

6

7

8

9

250
LacZ
98
64
50
36
30

eGFP

16

Figure 1: Dual expression in the Cascade™ Expression System.
LacZ and eGFP were cloned into pCHROMO and pCHROMO2, respectively, and integrated into TAP E. coli. Five hours after induction (2 mM
salicylate), the cells were lysed and co-purification was performed
using Ni-TED Silica, then run on a 4-20% Tris-Glycine gel.
Lane 1:
Protein standard (kDa)
Lane 2:
LysateLane 3:
Flow-throughLanes 4-5: Washes
Lanes 6-9: Elutions

Flexible formats
Ni-TED comes in a variety of formats tomeet your purification needs. For lowvolume elutions in small-scale protein
isolations, Ni-TED Spin Columns featurea silica-based membrane with a binding
capacity of 50 µg that is optimized for use
with proteins or peptides of < 100 kDa.
These Ni-TED Spin Columns are conveniently available in packages of 25 or 100 with orwithout Lysis, Wash and Elution Buffers. To
meet your large-scale needs, Ni-TED Silica
is available in prepackaged columns or inbulk for purifications from culture volumesof up to 100 ml. The Ni-TED silica resin willbind up to 3 mg of His-tagged protein pergram and is suitable for isolating proteinsof any size. For quick, efficient purification
you can count on, try Ni-TED today.

Product

FormatCatalog No.

Ni-TED™ Spin Columns

25 rxns
100 rxns

97025
97100

Ni-TED™ Spin Columns (w/solutions)

25 rxns
100 rxns

98025
98100

Ni-TED™ Silica (0.3 g resin in prepacked columns)

5 columns
20 columns

97005
97020

1g

97001

Ni-TED™ Silica

Toll Free — 1 877 222 9543

CONTACT INFO
US1914 Palomar Oaks Way, Suite 150
Carlsbad, CA 92008
Toll Free: 877 222 9543
Direct: 760 431 1263
Fax: 760 431 1351
Email: tech_service@activemotif.com

EUROPE
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Direct: +32 (0)2 653 0001
Fax: +32 (0)2 653 0050
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