NEB UK - New England Biolabs (UK)

NEB UK
Update
Spring 2008
NEW ENGLAND
® BioLabs
The latest innovation in restriction enzyme technology ...
High Fidelity Restriction
Enzymes
These new engineered enzymes have reduced star
activity and offer increased flexibility in reaction
setup, maximizing results under a wider range of
conditions.
Certain properties have been altered, including
buffer requirements and enzyme fidelity to produce
improved enzymes that will allow more flexibility
under suboptimal conditions.
HF Restriction Enzymes are purified to the same high
standards as our other restriction enzymes and are
available at the same low price. More on page 5.
NEW ENGLAND
® BioLabs
Iceland: a natIon Powered by
water
Gullfoss, or Golden Falls, on Iceland’s
White River is one of the most powerful
waterfalls in Europe. Sightseers venturing
near this spectacle are greeted with a roaring,
surging wall of water, 100 meters wide. “The
torrent is a mighty symphony that overwhelms
you,” notes Iceland’s president, Olafur Ragnar
Grimsson. “Swathed in spray, you feel …
renewed.” ... continued on page 2.
In this issue ...
New Products
ET SSB, DNA Gyrase, GpC
Methyltransferase ... page 2
Protein Markers: Special Offer
40% off our entire range of protein
markers page 3
Tips and Tricks
Choosing the optimal buffer for double
digests page 4
High Fidelity Restriction
Enzymes
New engineered restriction enzymes with
reduced star activity page 5
High Performance PCR
Phusion High-Fidelity Polymerase and
the new PIKO Thermal Cycler page 6
30% off CST Support Reagents
Cell Lysis Buffers, Markers and General
Reagents page 9
Antibodies for Autophagy
Autophagy has been linked with a
number of disease states - view CST’s
range of related antibodies page 10
FREE Timer ...
with all orders of the Quick Blunting
Kit page 8

NEW PRODUCTS
NEW PRODUCTS
2
NEW ET SSB (Single-Stranded
Binding Protein)
Enhance the specificity and speed of your PCR
reactions
ET SSB (Extreme Thermostable Single-Stranded DNA Binding Protein) enhances
PCR reactions by stabilising single-stranded DNA, thus preventing reannealing.
Isolated from a hyperthermophilic microorganism, this single-stranded DNA
binding protein remains fully active after incubation at 95°C for 60 minutes. Due
to its extreme thermostability, ET SSB can be used in applications that require
extremely high temperature conditions, such as thermocycling, nucleic acid
amplification and sequencing.
Advantages
❚ Increases PCR yield and specificity and polymerase processivity
❚ Increase the yield and processivity of RT during RT-PCR
❚ Ideal for challenging reactions, including multiplex PCR, long range and GC-rich
amplifi cation, and amplification through regions with strong secondary structure
❚ Active in any polymerase buffer
M 1 2 3 4 5 C
Figure 1: Improve multiplex PCR with ET SSB. By adding
ET SSB, specific amplification was accomplished in PCR
using one to five primer pairs in increasing order (lanes
1-5). Lane C is the control reaction using 5 primer pairs,
with no ET SSB included (control reaction). Lane M: 100 bp
DNA Ladder.
Ordering Information:
#M2401S 50 µg £106
Iceland: a natIon Powered by water
Given its mountainous terrain, frequent rain and abundance of ice and snow, Iceland is exceptionally well-suited for waterfalls and hosts many of Europe’s
largest. On this land, however, water doesn’t just drop from above; heated to the boiling point by volcanic magma below, it can burst through the earth’s surface
and shoot hundreds of feet into the air. Examples of such dramatic sprays can be found in Iceland’s Geysir Field, from which the word “geyser” originated.
In addition to reveling in the natural splendor of their waterfalls and geysers, the citizens of Iceland have also put their moving water to good use. Homes
and businesses are almost entirely heated by water in the form of geothermal energy that bubbles up from the interior. Except for the modest portion generated in
geothermal power plants, the vast majority of the nation’s electricity is generated by falling water, or hydroelectric power. All told, more than 95 percent of the heat
and electricity in Iceland comes from these two renewable sources.
The country still relies on imported oil to power its cars, boats and other transportation vehicles, which account for about 28 percent of the total energy
demand. But a campaign has begun to use Iceland’s most bountiful resource, water, to run the transportation sector as well. The plan is to tap the ample
hydropower reserves to convert water into hydrogen and oxygen by means of electrolysis. The hydrogen, in turn, would fuel the nation’s transportation vehicles.
As a first step, three buses (out of a total fleet of 80) now operate on hydrogen, and the world’s first hydrogen filling station opened in 2003. The goal, in time, is
to increase the number of such fueling stations (16 could cover the whole country), make all the buses hydrogen-powered and then convert the nation’s fishing
vessels and 180,000 cars.
M - + M ET SSB
Figure 2: ET SSB enhances long
range amplification. A PCR reaction
using primers specific for a 7.4kb
PCR product shows that the presence
of ET SSB enhances amplification
of the 7.4kb target, and reduces
amplification of non-specific bands.
Marker M is the 1 kb DNA Ladder
(NEB #N3232).
Developed and produced by BioHelix Corp., an
NEB-affiliated company.
The idea of hydrogen fuel is not new and was even discussed by visionary novelist Jules Verne in 1870. Yet Iceland is well-positioned to exploit it and, in so doing,
7.4 kb
could become the first country to have a fossil-free energy economy completely powered by water in various guises.
Hpy166II Br C 4
Recognition Site: GTN^NAC
NEW
R0616S 500 units 10,000 units/ml £51
R0616L 2,500 units 10,000 units/ml £204
GpC Methyltransferase
(M.CviPI)
M0227S 200 units 4,000 units/ml £48
M0227L 1,000 units 4,000 units/ml £192
The GC Methyltransferase, M.CviPI, methylates all
cytosine residues (C5) within the double-stranded
dinucleotide recognition sequence 5´...GC...3´.
Applications:
❚ Blocking restriction endonuclease cleavage
❚ Altering the physical properties of DNA
❚ Uniform [ 3 H]-labeling of DNA
Reagents Supplied:
GC Reaction Buffer (10X)
S-adenosylmethionine (SAM) (200X)
DNA Gyrase (E. coli)
M0306S 100 units 5,000 units/ml £53
M0306L 500 units 5,000 units/ml £212
DNA Gyrase (E. coli) is a Type II topoisomerase that
catalyzes the introduction of negative supercoils in
DNA in the presence of ATP. The gyrase holoenzyme
is a heterotetramer made up of 2 gyrA (97 kDa)
subunits and 2 gyrB (90 kDa) subunits.
Reagents Supplied:
DNA Gyrase (E. coli) Reaction Buffer
DNA Gyrase (E. coli) Substrate
Companion Product:
DNA Gyrase (E. coli) Substrate
NEW
NEW
N0471S 20 µg 1,000 µg/ml £51
A double-stranded covalently closed circular DNA
molecule produced by incubating supercoiled
pUC19 DNA with Topoisomerase I (E. coli).

ColorPlus Prestained
Protein Marker,
Broad Range
■ a mixture of purified proteins covalently
coupled to colored dye
■ resolves to 8 bands of even intensity
■ one orange and one green reference band
The covalent coupling of the dye to the proteins
affects their electrophoretic behavior in
SDS-PAGE gels relative to unstained proteins.
For precise molecular weight determinations,
use NEB's unstained Protein Markers (NEB
#P7702 or #P7703) in addition to the prestained
marker.
Protein Ladder,
(10–250 kDa)
■ a mixture of 12 recombinant, highly
purified proteins
■ clearly identifiable sharp bands from
10–250 kDa (SDS-PAGE stained with Coomassie
Brilliant Blue R-250)
■ for use as a precise size standard for SDS-
PAGE
■ 25 and 80 kDa bands have triple intensity
and serve as reference indicators
■ molecular weights confirmed by sequencing,
mass spectrometry and migration in a
Laemmli SDS-Page Tris-Glycine system.
Prestained
Protein Marker,
Broad Range
(6–175 kDa)
■ a mixture of purified proteins covalently
coupled to a blue dye
■ resolves to 8 bands when electrophoresed
■ protein concentrations are carefully
balanced for even intensity.
■ available in two formats
The covalent coupling of the dye to the proteins
affects their electrophoretic behavior in
SDS-PAGE gels relative to unstained proteins.
For precise molecular weight determinations,
use NEB's unstained Protein Markers (NEB
#P7701 or #P7703) in addition to the prestained
marker.
NEW
NEW
10-20%
SDS PAGE
10-20%
SDS PAGE
10-20%
SDS-PAGE
kDa
–175
– 83
– 62
– 47.5
– 32.5
– 20.5
– 16.5
– 6.5
kDa
– 250
– 150
– 100
– 80
– 60
– 50
– 40
– 30
– 25
– 20
– 15
– 10
kDa
– 175
– 83
– 62
– 47.5
– 32.5
– 20.5
– 16.5
– 6.5
40% OFF
Protein
Markers *
■
■
■
Convenience - Coloured band marker for easy gel
orientation.
Ordering Information:
ColorPlus Prestained Protein Marker, Broad Range
#P7709V 83 mini-gel lanes £53 £31.80
#P7709S 175 mini-gel lanes £78 £46.80
SPECIAL
OFFER
Speed - Prestained 1 tube formats for speed, quickly heat
prior to loading.
Accuracy - 3 tube formats of the pre- and unstained
protein markers allow for optimal results. 3 tube formats
consist of the protein molecular weight marker in a neutral
buffer, the loading buffer and reducing agents in separate tubes.
The 3 tube format allows the marker to be used in other gel
systems and to be stored safely; then immediately prior to use,
mixed with loading buffer and reducing agent.
#P7709L † 875 mini-gel lanes £322 £193 . 20
Protein Ladder, (10–250 kDa)
#P7703S 100 mini-gel lanes £54 £32.40
Prestained Protein Marker, Broad Range (6-175 kDa)
(Premixed Format)
#P7708V 83 mini-gel lanes £52 £31.20
#P7708S 175 mini-gel lanes £73 £43.80
#P7708L † 875 mini-gel lanes £292 £175.20
Also Available at 40% OFF:
Prestained Protein Marker, Broad Range (6-175 kDa)
(3 Tube Format)
#P7707S 125 mini-gel lanes £60 £36
#P7707L † 625 mini-gel lanes £240 £144
Protein Marker, Broad Range Premixed Format
#P7702S 150 mini-gel lanes £52 £31.20
#P7702L † 750 mini-gel lanes £208 £124.80
Protein Marker, Broad Range 3 Tube Format
#P7701S 100 mini-gel lanes £50 £30
#P7701L † 500 mini-gel lanes £200 £120
† #P7709L, #P7708L, #P7707L, #P7702L and #P7701L supplied as
5 packages of #P7709S, #P7708S, #P7707S, #P7702S and #P7701S
respectively.
* 40% discount is available to UK customers on the products listed
above until 30th April 2008. No other discounts apply.
NEW PRODUCTS
PROTEIN TOOLS 3
3

4
TITLE 4
TIPS & TRICKS
4
The NEBuffer System
For optimal restriction enzyme digests
NEB provides a colour-coded 10X NEBuffer with each restriction enzyme to ensure optimal (100%) activity.
Most of our enzymes are supplied with one of our four standard NEBuffers. Occasionally, an enzyme has
specific buffer requirements not met by one of the four standard NEBuffers, in which case the enzyme is
supplied with its own unique NEBuffer. As part of our ongoing committment to research, we are constantly
looking for ways to improve the production and efficiency of restriction enzymes. This may result in changes to
buffer requirements which has been the case recently for one of our most commonly used enzymes: BamHI.
BamHI B r C 3 b 6 n p
Cleaving a DNA substrate with two restriction
endonucleases simultaneously (double
digestion) is a common timesaving procedure.
Selecting the best NEBuffer to provide
reaction conditions that optimize enzyme
activity as well as avoid star activity associated
with some enzymes is an important
consideration. Each enzyme is supplied with
its optimal NEBuffer to ensure 100% activity.
NEBuffer compositions are listed on page
278 of our current catalogue and on the data
card sent with each enzyme. The Activity
Chart for Restriction Enzymes (see below to
request your free poster) rates the percentage
activity of each restriction endonuclease
in the four standard NEBuffers. The
NEBuffer that results in the most activity for
both enzymes should be used for the double
digest as long as star activity is not a factor.
Depending on an enzyme's activity rating
in a non-optimal NEBuffer, the number of
units of enzyme or incubation time of the
reaction may be adjusted to compensate for
a slower rate of cleavage.
If no single NEBuffer can be found to satisfy
the buffer requirements of both enzymes,
the reactions can be done sequentially.
First, cleave with the restriction endonuclease
that requires the lower salt reaction
conditions, then adjust the salt concentration
of the reaction (using a small volume of
a concentrated salt solution) to approximate
The New and
Updated
NEBuffer
Poster
BamHI is now supplied with NEBuffer 3
instead of a Unique Buffer following improvements
to the production of this enzyme
Setting up a Successful Double Digest
the reaction conditions of the second
restriction endonuclease. Add the second
enzyme and incubate to complete the second
reaction. Alternatively, a spin column
can be used to isolate the DNA prior to the
second reaction.
Performing double digests with enzymes
that are supplied with their own unique
NEBuffer and not one of the four standard
NEBuffers is also simple. In many
cases, double digests using any of these
enzymes with an enzyme that is supplied
with one of the four standard NEBuffers
can be done in the unique NEBuffer. This
will ensure that the enzyme with the more
specific buffer requirements will work
optimally. See page 284 of our current
catalogue for the most commonly used
restriction endonucleases’ activity ratings
in unique buffers. When using restriction
endonucleases in non-optimal NEBuffers,
more enzyme or longer digestion time may
be needed to compensate for the slower
rate of cleavage under those conditions.
Check the Activity Chart for Restriction Enzymes
to see how well the second enzyme
performs in the salt range of the unique
NEBuffer.
NEBuffer 1 2 3 4
% Activity 75 100 100 100
Double Digest Tips:
If BSA is a buffer requirement for either enzyme,
add it to the double digest reaction. BSA will
not inhibit any restriction enzyme.
The final concentration of glycerol in any
reaction should be less than 5% to minimize
the possibility of star activity. All our restriction
enzymes are supplied in 50% glycerol so the
total volume of enzyme in a reaction should
be a maximum of 10% of the total reaction
volume. An increase in total reaction volume
may be necessary.
Double digestion is not recommended for
certain enzyme combinations. In these cases a
sequential digest is required. These combinations
are indicated by the abbreviation "seq" in
the Double Digest table on the buffer poster.
Our data is based on 1–2 hour digests. Overnight
double digests should be avoided due to
possible star activity.
BsgI requires SAM. SAM as an additive does not
have a negative effect on the activity of other
enzymes.
For help choosing double digest conditions
try Double Digest Finder at www.neb.uk.com
This web-based tool can be used to suggest
conditions for double digestion using any two
NEB restriction enzymes.
The NEBuffer chart helps you select
the best conditions for double
digests, shows the optimal (supplied)
NEBuffer and approximate activity in
the four standard NEBuffers for each
enzyme.
To request a copy please email us:
bufferposter@uk.neb.com
or use our online literature request
form at: www.neb.uk.com

High Fidelity (HF) Restriction Enzymes
The latest innovation in restriction enzyme technology
As part of our ongoing commitment to the study and improvement
of restriction enzymes, we are pleased to introduce a line of High
Fidelity (HF) restriction enzymes. These engineered enzymes have
the same specificity as their established counterparts, however
certain properties have been altered, including buffer requirements
and enzyme fidelity. These modifications provide more flexibility in
setting up restriction enzyme digests. The overall goal of engineering
restriction enzymes is to provide improved enzymes that will allow
more flexibility with respect to reaction volume, incubation time and
buffer compatibility. Each of these enzymes has been purified to the
same high standards as our other restriction enzymes, and are available
at the same low price.
The introductory selection of engineered restriction enzymes offers
the benefit of reduced star activity. Star activity, or relaxed specificity,
is an intrinsic property of restriction enzymes. Most restriction
enzymes will not exhibit star activity when used under the
recommended reaction conditions. However, for enzymes that have
reported star activity, extra caution must be taken to set up reactions
under the recommended conditions to avoid unwanted cleavage. Different techniques
such as cloning, genotyping, mutational analysis, mapping, probe preparation, sequencing
and methylation detection employ a wide range of reaction conditions and require
the use of enzymes under suboptimal conditions. These new high fidelity (HF) enzymes
will offer increased flexibility to reaction setup, maximizing results under a wider range
of conditions.
In addition to reduced star activity, all of these engineered enzymes work optimally in
NEBuffer 4, which has the the highest level of enzymes compatibility, and may simplify
double digest reactions. They are also Time-Saver qualified, and will digest substrate
DNA in five minutes.
In order to distinguish these engineered enzymes, the letters –HF have been added to
the restriction enzyme name. An icon ( E ) designating that the enzyme has been engineered
will appear with the product entry, on the datacard and on the website. In addition,
icons for the enhanced properties (such as reduced star activity, R ) that these new
enzymes possess will also be included. These enzymes will be packaged with a purple
cap to distinguish them from our existing restriction enzymes.
Five HF enzymes are currently available with more to follow.
Ordering Information:
NheI-HF E R
#R3131S 1,000 units 20,000 units/ml £44
PvuII-HF E R
#R3151S 5,000 units 20,000 units/ml £45
SalI-HF E R
#R3138S 2,000 units 20,000 units/ml £40
ScaI-HF E R
#R3122S 1,000 units 20,000 units/ml £45
SphI-HF E R
#R3182S 500 units 20,000 units/ml £48
same low
price as
standard
enzymes
ScaI-HF
M 4 3 4 NEBuffer
ScaI
ScaI is one of the most frequently reported enzymes to
exhibit star activity (unwanted cleavage). While star activity is
observed with ScaI in NEBuffer 3 (supplied buffer) as well as
NEBuffer 4, star activity is significantly reduced with ScaI-HF.
20 μl reactions were set up containing 2 μl of enzyme and
incubated for 1 hour. Conditions were designed to simulate
those obtained when a double digest is performed in a 20 μl
reaction. Marker M is the 1 kb DNA Ladder (NEB #N3232).
New England Biolabs ...
for the highest quality Restriction Enzymes.
Manufactured and quality controlled by NEB – our restriction
enzymes aren’t made by anyone else.
*
*
*
*
NEW
unwanted cleavage
TITLE 5
PROTEIN LATEST NEWS: TOOLSRESTRICTION ENZYMES
3 5
5

TITLE 6 4
POLYMERASES & AMPLIFICATION HIGH PERFORMANCE TECHNOLOGIES PCR
6
64
High Performance PCR
Finnzymes has deconstructed the PCR process and created a tripartite solution that
improves nearly every measurable aspect of PCR. It combines highly processive
proofreading Phusion DNA Polymerases, high-speed Piko Thermal Cyclers, and ultrathin
walled UTW tubes and plates. Each component alone, is best in class; together they
offer synergies that propel PCR to unbeatable performance.
Advantages of High Performance PCR
Speed – Significantly faster than any other combination
Fidelity – Superior accuracy over Taq and Pfu based systems
Yield – Higher efficiency amplification results in more product
Specificity – Reduced levels of primer-dimers and false-primed
products
Speed - Fastest enzyme,
fastest instrument, fastest tubes
To save valuable research time, each component of the High Performance
PCR solution is designed to expedite PCR protocols. Phusion DNA
Polymerases are highly processive, due to a DNA-binding domain fused
to the polymerase. The result is extreme reaction speed and robustness.
The Phusion Flash PCR Master Mix requires only 15 seconds or less to
extend a 1kb target. Clever designing enables the Piko Thermal Cycler
to complete a 25-cycle PCR protocol in as little as 10 minutes. This
speed is achieved by a fast ramp rate and an incredible settling time of
less than 1 second, which allows for 0 second holds at annealing and
denaturing steps. The vessels (UTW tubes and plates), have extremely
thin walls where the tubes contact the cycler block. This results in less
thermal resistance and rapid heat transfer from the block to the reaction
0.7 kb
1.3 kb
2.0 kb
(min) 0 20 40 60 80 100
High Performance PCR
Conventional PCR with Taq
Comparison of total cycling times in amplification of genomic DNA fragments between
0.7-2.0 kb. The increased processivity of Phusion DNA Polymerases allows extremely short
cycling times. Additionally, annealing temperatures used are 5°– 8°C above conventional
PCR. Both of these shave valuable seconds off each cycle and make 2-step protocols easily
achievable. The extraordinary short hold times are due to the temperature stabilizing effect
of the Piko Thermal Cyclers and UTW vessels, minimizing the time required to achieve
the target temperature.
Yield – Amplify with confidence
High speed PCR is often fickle – variable yields depend upon the purity
of template and the need for tedious reaction optimization. This is not
true for our integrated solution. Phusion technology offers several distinct
advantages. First, we allow for high efficiency amplification under the
most demanding conditions. Phusion DNA Polymerases have a very
broad window for optimization that allows them to work robustly with
common inhibitors of PCR (such as blood) and in varying reaction
conditions. Second, the extreme stability of Phusion DNA Polymerases
at high temperatures, combined with the ultra-quick settling time of the
Piko Thermal Cyclers, results in consistently abundant yields of specific
product – even with prolonged PCR protocols.
High Performance
PCR
Conventional
PCR with Taq
Conventional
PCR with Pfu
20 min 63 min 99 min
High Performance PCR - higher yields in shorter time. Amplification of a
0.7 kb fragment from human β-glucuronidase gene with Finnzymes’ High Performance
PCR (using Phusion Flash High Fidelity DNA Polymerase Master Mix) versus conventional
PCR protocols with Taq or Pfu DNA polymerases. After PCR, equal aliquots of samples
(duplicates of each) were run on a 1.5 % agarose gel.
mixture. Fidelity – We challenge you to find
an error
At the core of our unique polymerases is a cleverly engineered proofreading
enzyme with enhanced DNA-binding activity and processivity. The
Phusion technology improves the fidelity of the polymerase, such that
Phusion High-Fidelity DNA Polymerases have the lowest error rates of
any polymerases available. The error rate of Phusion DNA Polymerases
is as low as 4.4 x 10 -7 , which is more than 50-fold better than that of
Taq DNA polymerase. High Performance PCR may be used in routine
applications or with protocols requiring the highest accuracy.
DNA Polymerase Fidelity Products with
value errors (%)
Phusion High-Fidelity DNA Polymerase 4.40 x10 -07 1.32 %
Pfu DNA polymerase 2.80 x10 -06 8.40 %
DNA polymerase from T. kodakaraensis 3.32 x10 -06 9.96 %
Taq DNA polymerase 2.28 x10 -05 68.4 %
High Performance PCR guarantees extreme fidelity. Calculated percentage (%) of PCR
products (1 kb) having an error after a 30-cycle PCR reaction. 99 out of 100 fragments
amplified with High Performance PCR have no errors, whereas Taq DNA polymerase
produces only 32 error-free fragments out of 100.
Specificity – One lane, one band
Primer-dimers and false-primed products are not only a nuisance, but
may mask the correct target amplicon. Our integrated solution combats
this by speeding and heating the reaction. The Piko Thermal Cycler uses
fast ramping and short annealing times to reduce the effects of spurious
amplification by minimising false priming. These unwanted products are
diminished by Phusion DNA Polymerase’s ability to dramatically raise
annealing temperatures as compared with conventional PCR. To further
improve specificity in PCR, Phusion DNA Polymerases are also available
as hot start enzymes utilizing “zero-time reactivation.”

High Performance PCR
THE INTEGRATED SOLUTION
Phusion High-Fidelity DNA
Polymerases
Phusion DNA Polymerases, with a combination of extreme fidelity,
unparalleled speed and robustness, guarantee PCR performance that no
other enzyme can match.
Piko Thermal Cyclers
Phusion Technology
Phusion technology fuses a novel
pyrococcus-like proofreading
polymerase with a DNA binding
domain. The Phusion technology
delivers the highest fidelity with
shorter extension times and
minimal optimization.
Advantages
Accuracy – DNA Polymerases
with the highest fidelity
Speed – Extremely short
extension times (as little as 15
s/kb or less)
Robustness – Minimized
reaction failures and
optimization
High yields – Increased product
levels
Specificity –Reduced levels of
non-specific amplification
Piko Thermal Cyclers offer high thermal performance in a tiny
footprint.
Piko Technology
At the heart of the Piko Thermal
Cycler lies a unique heat pump.
Designed to deliver bridled power,
it results in industry-leading
thermal uniformity, accuracy,
ramp rates and settling times. This
not only allows for PCR protocols
in as little as 10 minutes, but also
assures consistent results from
well-to-well and instrument-toinstrument.
Advantages
Half the size – Smallest footprint
at 16 cm x 17 cm
Twice the speed – PCR protocols
in as little as 10 minutes
Superior thermal uniformity
– Robust and consistent results
Phusion and Piko are trademarks of Finnzymes Oy or its affiliates. UTW is a trademark of BioInnovations
Oy.
Notice to Purchaser of Piko Thermal Cyclers: Purchase of this instrument conveys a limited
non-transferable immunity from suit for the purchaser’s own internal research and development
and applied fields other than human in vitro diagnostics under non-real-time thermal cycler
patents of Applera Corporation. The quality system of Finnzymes Oy is certified according to
standard SFS-EN ISO9001:2000
UTW Tubes and Plates
Finnzymes’ ultra-thin walled (UTW ) vessels are designed for High
Performance PCR applications. Their unique features allow for extremely
short protocol times with consistent performance.
Ordering Information:
Phusion High Fidelity DNA Polymerase
#F-530S/L 100/500 units £65/£295
Phusion (Hot Start) High Fidelity DNA Polymerase
#F-540S/L 100/500 units £78/£354
Phusion Flash High Fidelity DNA Polymerase Master Mix
#F-548S/L 100/500 20µl reactions £54/£216
Phusion High Fidelity DNA Polymerase Master Mix with HF Buffer
#F-531S/L 100/500 50µl reactions £106/£424
Phusion High Fidelity DNA Polymerase Master Mix with GC Buffer
#F-532S/L 100/500 50µl reactions £106/£424
Piko Thermal Cycler, 24-Well System
#TCP0024 Each £2,200
Piko Thermal Cycler, 96-Well System
#TCP0096 Each £2,935
24-Well Slidetitre Plate (Clear)*
#SPL0240 box of 200 £200
96-Well Slidetitre Plate (Clear)*
#SPL0960 box of 200 £250
UTW Individual Tube (Clear)*
#TUC0010 bag of 1,000 £46
UTW 8-Tube Strips (Clear)*
UTW Technology
Ultra-thin walled PCR tubes and
plates are manufactured utilizing
a patent-pending process for
molding polypropylene vessels
with walls that are a mere 150
microns thick. Thinner walls
offer less thermal resistance to
heat transfer from the cycler
block to the sample within
the tube. This allows for much
greater cycling speeds.
Advantages
High thermal conductivity
– Half the thickness of
conventional tubes
Uniform thinness – Equal
performance in every well
Reagent savings – Reaction
volumes down to 5 µl
#TUC0080 bag of 125 £42
* For full range of plasticware please visit www.neb.uk.com
NEW
HIGH PERFORMANCE PCR
7 7

CLONING
8
Quick Blunting Kit
For efficient blunt-end ligations
Optimize your blunt-end cloning reactions with the Quick Blunting Kit from NEB.
This kit provides a fast and convenient method for preparing sheared, nebulized or
restriction enzyme-digested DNA for blunt-ended ligation into a plasmid, cosmid,
fosmid or BAC vector. In addition, it can also be used to prepare PCR products
generated using non-phosphorylated primers for efficient blunt-end cloning.
Advantages of Quick Blunting Kit
❚ Fast – Restriction enzyme-digested DNA
blunted in less than 30 minutes
❚ Convenient – Reactions are performed at
room temperature in a ready-to-use mix
❚ Flexible – Suitable for restriction enzymedigested
DNA, sheared or nebulized DNA and
PCR products
Ordering Information:
Quick Blunting Kit plus FREE Timer
#E1201S 20 Reactions £60
#E1201L 100 Reactions £240
Quick Blunting & Quick Ligation Kit
#E0542S 20 Blunting Reactions +
30 Ligation Reactions
Restriction Enzyme-
Digested DNA
5´
5´
5´
5´
5´
5´
5´
A 5´
A 5´
5´
5´P
OH
Blunted
Dephosphorylated
Vector
or Purified PCR or
Product
Quick Blunting
OH
P5´
Ligation
Transform + Plate
Sheared
DNA
The Quick Blunting Kit facilitates blunt-end cloning reactions.
FREE Timer with
either of these kits!
Available until 30th June 2008.
All the advantages of the Quick Blunting Kit PLUS
ligations in just 5 minutes at room temperature
This bundle of complementary kits comes in two sizes providing enough
reagents for 20 or 100 reactions and and is more economical than buying
two kits separately.
Advantages of Quick Ligation
❚ Fast – 5 minutes for cohesive or blunt ends
❚ Convenient – ligation performed at room temperature
❚ Flexible – suitable for all common ligation reactions
Ordering Information:
Quick Blunting & Quick Ligation Kit plus FREE Timer
#E0542L 100 Blunting Reactions +
150 Ligation Reactions
The Quick Ligation Kit may also be purchased separately:
#M2200S, 30 Reactions, £80; #M2200L, 150 Reactions, £320
£120 Save £20 compared to buying
kits separately
£480 Save £60 compared to buying
kits separately
Using the Quick Ligation Kit protocol, blunt
and cohesive inserts were ligated into Litmus
28 vector cut with either EcoR V (blunt) or
Hind III (cohesive). Ligation products were
transformed into chemically competent E. coli
DH-5acells.

SPECIAL OFFER
CST Lysis Buffers, Markers
and Support Reagents
For consistently great results choose the same reagents
that CST use for Quality Control testing
Lysis Buffers Normal
Price
Cell Lysis Buffer (10X)
#9803 15 ml £36 £25.20
ü for lysis of adherent and non-adherent cells – simply
add 1 mM PMSF immediately before use
ü 10x buffer can be used for lysis of tissue samples
(with homogenization)
ü used to lyse cells under nondenaturing conditions
ü 10x buffer can be kept at 4°C for 1-2 weeks (or at -20°C
for longer periods)
ü additional protease inhibitors can be added
1X concentration: 20 mM Tris-HCl (pH 7.5)
150 mM NaCl
1 mM Na 2 EDTA
1 mM EGTA
1% Triton
2.5 mM sodium pyrophosphate
1 mM beta-glycerophosphate
1 mM Na 3 VO 4
1 mg/ml leupeptin
RIPA Buffer (10X)
#9806 15 ml £36 £25.20
ü for lysis of adherent and non-adherent cells
– simply add 1 mM PMSF immediately before use
ü 10x buffer can be used for lysis of tissue samples
(with homogenization)
ü used to lyse cells under nondenaturing conditions
ü 10x buffer can be kept at 4°C for 1-2 weeks (or at
-20°C for longer periods)
ü additional protease inhibitors can be added
1X concentration: 20 mM Tris-HCl (pH 7.5)
150 mM NaCl
1 mM Na 2 EDTA
1 mM EGTA
1% NP-40
1% sodium deoxycholate
2.5 mM sodium pyrophosphate
1 mM beta-glycerophosphate
1 mM Na 3 VO 4
1 mg/ml leupeptin
30%
OFF *
RIPA Buffer (10X) #9806 15 ml £36 £25.20
Cell Lysis Buffer (10X) #9803 15 ml £36 £25.20
CHAPS Cell Extract Buffer (10X) #9852 5 ml £36 £25.20
Protein Markers
Biotinylated Protein Ladder Detection Pack #7727S
#7727L
Prestained Protein Marker, Broad Range (Premixed Format) #7720S
#7720L
Loading Buffers
65 mini-blots
325 mini-blots
70 lanes
350 lanes
Blue Loading Buffer Pack #7722 8 ml £14 £9.80
Red Loading Buffer Pack #7723 8 ml £14 £9.80
General Support Reagents
CST Cell Lysis Buffers – validated with CST antibodies to guarantee reproducible results
£36
£144
£62
£245
Offer
Price
£25.20
£100.80
£43.40
£171.50
BSA #9998 50 grams £144 £100.80
Nonfat Dry Milk #9999 250 grams £36 £25.20
Phosphate Buffered Saline (PBS-20X) #9808 1000 ml £54 £37.80
Phosphate Buffered Saline with Tween 20 (PBST-20X) #9809 1000 ml £62 £43.40
Tris Buffered Saline with Tween 20 (TBST - 10X) #9997 1000 ml £36 £25.20
ELISA Reagents
STOP Solution #7002S
#7002L
TMB Substrate #7004S
#7004L
Kinase Assay Reagents
100 ml (10 x 96 well plates)
500 ml (50 x 96 well plates)
100 ml (10 x 96 well plates)
500 ml (50 x 96 well plates)
£20
£70
£36
£144
£14
£49
£25.20
£100.80
ATP 10mM #9804 1 ml (100 Assays, 96 Well Format)) £36 £25.20
Kinase Buffer (10X) #9802 15 ml £36 £25.20
* 30% discount is available to UK customers on the products listed above until 30th April 2008. No other discounts apply.
Chaps Cell Extract Buffer (10X)
#9852 5 ml £36 £25.20
ü for lysis of cells under denaturing conditions – simply
add 5 mM DTT (200X conc. included) and 1 mM PMSF
immediately before use
ü can be used under nondenaturing conditions
ü for the preparation of cytoplasmic cell lysates to be
used with CST Caspase signaling pathway antibodies
1X concentration: 50 mM Pipes/HCI (pH 6.5)
2 mM EDTA
0.1% Chaps
20 mg/ml Leupeptin
10 mg/ml Pepstatin A
10 mg/ml Aprotinin
For use
with
Caspase
Antibodies
Cell Signaling Technology News 9

Cell Signaling Technology News
10
Autophagy
Autophagy is a catabolic process that results in the autophagosomic-lysosomal
degradation of bulk cytoplasmic contents.
Autophagy is generally activated by conditions of nutrient deprivation
but has also been associated physiological processes
such as development, differentiation, neurodegenerative diseases,
infection and cancer. The kinase mTOR is a critical regulator
of autophagy induction. Upon growth-factor mediated activation
of PI3K/Akt and MAP kinase signaling, activated mTOR
promotes cell growth and protein translation, and suppresses
autophagy; mTOR is negatively regulated by AMPK and p53 signaling
pathways, which promote autophagy.
The molecular machinery responsible for autophagy was largely
discovered in yeast and are referred to as autophagy-related
(Atg) genes. Autophagy is induced by a class III PI3 kinase lipidkinase
complex that includes the tumor suppressor Beclin -1 (Atg
6) as an essential component. Two widely conserved ubiquitinlike
conjugation systems, LC3 (Atg8)-phosphatdylethanolamine
and Atg12-Atg5 function in autophagosomal vesicle formation.
Autophagy and apoptosis are connected both positively and
negatively. During nutrient deficiency, autophagy functions as
a pro-survival mechanism; however, excessive autophagy leads
to autophagic cell death, a process morphologically distinct
from apoptosis. Despite this morphological difference, extensive
cross-talk occurs between autophagy and apoptosis. Several proapoptotic
signals, such as TNF, TRAIL and FADD, also induce autophagy
and pro-survival signaling through the PI3K/Akt/mTOR
pathway suppresses autophagy. Additionally, Bcl-2 binds Beclin-1
to inhibit Beclin-1-dependent autophagy, thereby functioning
both as a pro-survival and as an anti-autophagic regulator.
Precursor LC3B (dark red) is cleaved by Atg4 (yellow), then further processed by Atg7 and Atg3 (not shown) to produce a lipidated form (bright red) that becomes
associated with autophagosomal membranes (background). Free Atg12 (orange) is conjugated to Atg5 (brown) by a ubiquitin-like interaction; Atg12+5 then bind Atg16
(yellow-brown). This complex is involved in formation of the autophagosome membrane, possibly acting as a scaffold for membrane extension.

Light chain 3 (LC3), which can serve as a marker for autophagy, was originally identified as a
subunit of microtubule-associated proteins 1A and 1B (termed MAP1LC3) and was subsequently found to
be a homologue of the yeast protein Atg8 critical for autophagy. Three human isoforms of LC3 - LC3A,
LC3B and LC3C - undergo post-translational modifications during autophagy. LC3 is first cleaved at the
carboxy terminus immediately following synthesis to yield a cytosolic form LC3-I. During autophagy LC3-I
is converted to LC3-II through lipidation by a ubiquitin-like system involving Atg7 and Atg3 which results
in the association of LC3-II with autophagic vesicles. The presence of LC3 in autophagosomes as well as
the conversion of LC3 to the lower migrating form LC3-II are used as indicators of autophagy.
LC3B Antibody #2775
Confocal IF analysis of HeLa cells, untreated (left) or chloroquinetreated
(right), using #2775 (green). Actin filaments have been
labeled with Alexa Fluor ® 555 phalloidin (red). Blue = DRAQ5
fluorescent DNA dye.
Atg12 Antibody (Human Specific) #2010
Applications Reactivity
kDa
100
80
Confocal IF analysis of HCT-116 cells, untreated (left) or chloroquine-treated
(right), using #2010 (green). Blue = DRAQ5 fluorescent DNA dye.
Autophagy Signaling
60
50
40
30
20
HCT-116
THP-1
SR
PANC-1
Atg12-Atg5
free Atg12
WB analysis of
extracts from
various cell lines
using #2010.
In mammalian cells, mTOR integrates signals from nutrients and growth
factors to control regulation of cell growth and proliferation. mTOR is
activated by PI3K/Akt and MAP kinase signaling as well as amino acid
levels and inhibited by AMPK and p53 signaling, all of which integrate
the energy status and genotoxic stress levels within the cell. mTOR also
functions as major sensor in autophagy signaling. Activation of mTOR
promotes protein synthesis and inhibits autophagy. In yeast, the Atg1
Ser/Thr kinase complex functions downstream of mTOR to regulate
various steps of autophagosome formation. However, the role of Atg1 in
higher organisms has yet to be elucidated.
For a complete listing of CST antibodies, kits and related reagents
for the study of mTOR signaling please go to www.neb.uk.com.
Product
Code
Apoptosis
Phagophore
Atg7
Bcl-2
Atg10
Pack
Size Price
Beclin-1 Antibody W, IP, IF-IC H, M, R #3738 100 ml £144
New LC3B Antibody W, IF-IC H, M, R, (Mk, C, B, X) #2775 100 ml £144
New Atg5 Antibody W,IP H, (Mk) #2630 100 ml £144
New Atg7 Antibody W H, M, R, (Mk) #2631 100 ml £144
New Atg12 Antibody (Human Specific) W, IP, IF-IC H #2010 100 ml £144
New Atg12 Antibody (Mouse Specific) W, IP, IF-IC M #2011 100 ml £144
New PI3K Class III Antibody W, IP H, M, R #3811 100 ml £144
New BNIP3 Antibody (Rodent Specific) W, IP H, M, R #2775 100 ml £144
VPS15
PI3K III
Atg12
Atg5
Atg16
PI3K/Akt
Signaling
AMPK
Signaling p53/Genotoxic
Amino Acids
Beclin1
Atg7
Atg4
Atg3
mTOR (7C10) Rabbit mAb #2983
IHC analysis of paraffin-embedded
human breast carcinoma, showing
cytoplasmic localization using #2983.
LC3-I
Autophagy
Induction
GL
Ser2481
Ser2448
P P
Raptor
Membrane
Nucleation
Sequestration
LC3-II
Atg1
P
mTOR
PRAS40
P
Autophagosome
Fusion
Lysosome
MAPK/Erk1/2
Signaling
Stress
Autophagolysosome
Formation of the autophagosome involves a ubiquitin-like
conjugation system in which Atg12 is covalently bound to Atg5
and targeted to autophagic vesicles. This conjugation reaction
is mediated by the ubiquitin-E1-like enzyme Atg7 and the
E2-like enzyme Atg10.
Atg12 Antibody (Mouse Specific) #2011
Confocal IF analysis of NIH/3T3 cells, untreated (left)
or chloroquine-treated (right), using #2011 (green).
Actin filaments have been labeled with Alexa Fluor ®
555 phalloidin (red). Blue = DRAQ5 fluorescent
DNA dye.
To view the full range of CST
antibodies please visit
www.neb.uk.com
Cell Signaling Technology News
11

ELECTROCOMPETENT STRAINS
NOW AVAILABLE
Competent Cells from New England Biolabs
NEW STRAINS, NEW FORMATS, NEW CHOICES
At NEB, we make transformation as simple as selecting the best strain for your
experiment. Our expanded line of competent cells includes a variety of strains
for cloning and expression, as well as strains with unique properties (see chart).
For added convenience, we offer a choice of efficiencies, formats and customized
packaging. Choose the superior performance and quality of competent cells from
New England Biolabs.
Advantages:
■ Extremely high transformation efficiencies
■ Phage T1 resistance (fhuA2) preserves clone integrity
■ Choice of protocols: high efficiency or 5 minute transformation
■ Nonspecific endonuclease activity eliminated, resulting in highest
quality plasmid preparations
■ Express difficult or toxic proteins with T7 Express strains containing
lacI q and/or a novel lysY variant
■ Obtain colonies faster than any other commercial strain with NEB Turbo
■ SOC Outgrowth Media and pUC19 Control Plasmid included
■ Free of animal products
For more information information and our international please visit distribution www.neb.uk.com
network, please visit www.neb.com
For a copy of our new Competent Cell Brochure, please visit www.neb.com/literaturerequest
New England Biolabs (UK) Ltd, Knowl Piece, Wilbury Way, Hitchin, Herts SG4 0TY
Tel:
New
0800
England
318486
Biolabs Inc.
Fax:
240 County
0800
Road,
435682
Ipswich, MA 01938
info@uk.neb.com
USA 1-800-NEB-LABS Tel.
www.neb.uk.com
(978) 927-5054 Fax (978) 921-1350 info@neb.com
Canada Tel. (800) 387-1095 info@ca.neb.com • China Tel. 010-82378266 beijing@neb-china.com • Germany Tel. 0800/246 5227 info@de.neb.com
Japan Tel. +81 (0)3 5669 6191 info@neb-japan.com • UK Tel. (0800) 318486 info@uk.neb.com
© 2008 New England Biolabs (UK) Ltd
N E W
E N G L A N D
B I O L A B S
some things are worth waiting for.
Cloning strain characteristics Strain NEB #
Obtain colonies faster than any other NEB Turbo Competent E. coli * C2984H/I
commercial strain (6.5 hours)
Versatile cloning strain NEB 5-alpha Competent E. coli † * C2987H/I
Cloning of toxic genes NEB 5-alpha F´l q Competent E. coli C2992H/I
Cloning of large plasmids and BACs NEB 10-beta Competent E. coli * C3019H/I
Growth of unmethylated plasmids dam – /dcm – Competent E. coli C2925H/I
Expression strain characteristics Strain NEB #
Most popular non-T7 protein NEB Express Competent E. coli C2523H/I
expression strain
Added control of IPTG induced NEB Express l q Competent E. coli C3037H/I
expression with non-T7 plasmids
Most popular T7 protein expression strain T7 Express Competent E. coli C2566H/I
Reduced basal expression T7 Express l q Competent E. coli C3016H/I
Tight control of protein expression T7 Express lysY Competent E. coli C3010H/I
by inhibition of T7 RNA Polymerase
Highest level of protein expression control T7 Express lysY/l q Competent E. coli C3013H/I
For crystallography experiments/SeMet T7 Express Crystal Competent E. coli C3022H/I
labeling
† Available as subcloning efficiency * Available as electrocompetent cells
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