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GE Healthcare
Protein and nucleic acid
sample prep.
Get it right from the start.
Why preparation
is everything
Getting started can be the hardest thing to do
GE Healthcare understands the challenges that need to be met in life science
research. For over 50 years we have been developing analytical techniques
and biomolecule preparation chemistry. Today, we are continuously working
with our customers to optimize the entire workflow. That’s why our sample
preparation products have been designed with specific analytical techniques
in focus and with these principles in mind:
• Preparing protein, DNA and RNA samples is just as critical
as the analysis step
• Reducing errors and increasing reproducibility at the preparation
stage ensures the best results
• Genomic and protein research workflows involve combinations
of biomolecules and are becoming more complex
• Identifying the right product for each stage of the research process
makes it easier to get great results
We have simplified the process and made it easier for users to get started
by providing the right product for each stage of the process.
A good start leads to a great finish
The illustra™ platform for nucleic acid preparation and the Trap platform for
protein preparation provide high standards of quality. The straightforward
protocols generate reproducible results with exceptional yield and purity.
The illustra platform provides simplicity through reliable kit design and readyto-use components, and the Trap platform provides optimized protocols
for specific proteins and analytical techniques through a range of formats,
buffers, media, and optimization guides.
This sample preparation workflow and product selection guide are designed to
help you choose the best solutions for your research. To find out more about the
applications see the corresponding pages in the brochure or visit our website:
www.gelifesciences.com/sampleprep
Regardless of your sample source, purification target or downstream
application, illustra and Trap products enable you to get a great start
for an excellent finish.
DNA/RNA
illustra nucleic
acid expertise
Purify. Amplify. Simplify.
Fast Flow media
illustra: speed with the quality you expect
Proper sample preparation is critical to achieving consistent
and reproducible results. illustra products have been developed to simplify the sample preparation process for genomic
research. illustra nucleic acid purification and amplification
products embody GE Healthcare’s expertise, incorporating
novel design components such as:
illustra Fast Flow media is specifically designed for high
absorption and fast purification of biomolecules. It captures
DNA quickly and efficiently and its ability to function at high
salt concentrations enables nucleic acids to be eluted with
a high degree of purity.
Nucleic acid purification and amplification just got easier with
illustra. From RNA, genomic DNA and plasmid DNA purification
to PCR, plasmid and whole genome amplification, illustra
products make it simpler and faster to produce quality results
in your lab. illustra delivers optimal yield and purity, ensuring
superior quality for use in downstream analysis. It can be used
with a wide range of sample sources, blood, tissue, cells and
bacteria. With more than 20 years of experience in nucleic
acid research, GE Healthcare knows how to get your experiments right from the start.
Phi29 DNA polymerase-based amplification technology
Highly reproducible Ready-To-Go technology
Our Ready-To-Go™ (RTG) technology ensures reproducibility
by stabilizing temperature-sensitive protein and nucleic acid
molecules into a single-dose bead which is stable at room
temperature. Each bead contains all the components, except
user defined template and primers, for a particular molecular
biology reaction, such as PCR, RT-PCR or labeling. RTG reduces
the risks of contamination and handling errors. RTG also
saves on shipping, freezer storage and reagent preparation.
Our Phi29 technology provides 100-fold higher fidelity in
amplification compared to Taq DNA Polymerase. The Phi29
DNA polymerase enzyme works quickly, and produces
stronger strand displacement and better 3’–5’ exonuclease
proofreading activity. The easy-to-use isothermal amplification method produces microgram quantities of DNA
from nanogram amounts of starting material.
High quality:
Speed:
illustra tissue and cells genomicPrep Midi Flow Kit delivers
genomic DNA of a larger size and higher quality than
Genomic-tip 100/G from Qiagen. Genomic DNA was
isolated from 80 mg of rat liver using each kit according
to manufacturers’ instructions. See data file 28-9162-80
for comparison details.
illustra tissue and cells genomicPrep Mini Spin Kit
illustra = 90 min prep
0
45
0
15
45
30
15
30
QIAamp™ DNA Mini Kit = 200 min prep
0
illustra Hot Start Mix RTG for PCR requiring high
specificity of amplification.
Schematic diagram of the amplification process with the
GenomiPhi™ amplification method. Random hexamer
primers anneal to the template DNA at multiple sites.
Phi29 DNA polymerase initiates replication at multiple sites
on the denatured linear DNA simultaneously. As synthesis
proceeds, strand displacement of complementary DNA
generates new single-stranded DNA. The subsequent
priming and strand displacement replication of this DNA
results in the formation of double-stranded DNA.
45
0
30
15
45
0
15
45
30
0
30
15
45
15
30
The illustra tissue and cells genomicPrep Mini Spin Kit requires
considerably less hands-on time than the QIAamp DNA Mini Kit
from Qiagen.
Cloning and
Sequencing
Cloning and
Sequencing
Sample preparation for
cloning and sequencing
Plasmid DNA preparation
50% reduction in protocol time
PCR
Convenient bead format, robust PCR results
Cloning and sequencing require intensive and routine
preparation of pure plasmid DNA, fosmids and bacterial
artificial chromosomes (BAC). illustra kits are designed
to speed up and significantly simplify the process for
DNA sample prep, downstream PCR and clean-up.
illustra plasmidPrep Mini Spin Kit yields highly pure plasmid in
less than 10 min with minimal changes in pipetting volume.
The illustra plasmidPrep Midi Flow Kit increases DNA yield
with excellent purity and low endotoxin levels.
Today, PCR is such a commonly used technique that methods
have to be convenient, robust and high performing. Beadbased technology provides all these benefits.
illustra PuReTaq RTG PCR Beads provide the convenience
of easy reaction setup and storage. The beads are provided
in either 0.2 or 0.5 ml tubes that are compatible with most
thermal cyclers; you just need to add template and primers.
In a comparative study using the same assays, templates
and instruments, illustra PuReTaq RTG PCR Beads showed
better performance against AmpliTaq Gold™ PCR Master Mix.
• illustra PuReTaq™ Ready-To-Go PCR Beads: ambient
temperature-stable PCR kit with PuReTaq DNA polymerase
and other high-purity reagents for reliable and robust
performance in both endpoint and real-time fluorescence
based PCR amplification
• illustra plasmidPrep Mini Spin Kit: high-quality plasmid
DNA purification in less than 10 min
• illustra plasmidPrep Midi Flow Kit: excellent purity and
low endotoxin level with increased column capacity that
extends the midi kit’s capability to maxi range input
• illustra TempliPhi™ 100/500 Amplification Kit: culturefree and simple plasmid DNA preparation for improved
sequencing results
Culture-free plasmid DNA, low hands-on time, high
sequencing success
illustra TempliPhi DNA Amplification Kits are used to
prepare DNA directly from plasmid or fosmid glycerol
stocks or colonies, thus eliminating overnight culture
preparation, reducing hands-on time, and improving
sequencing success and read length.
illustra plasmidPrep Mini Spin Kit delivers high-quality
plasmid DNA in half the time of QIAprep™ Spin Miniprep Kit.
Results obtained using three different researchers isolating
nine different plasmids. Plus (+) refers to the inclusion of an
optional nuclease removal step and (-) denotes its omission.
See data file 28-9075-92 for comparison details.
The blue real-time PCR growth curves are results obtained
using PuReTaq Ready-To-Go PCR Beads; red growth curves
are for AmpliTaq Gold PCR Master Mix. All curves were
obtained under identical conditions, with E. coli DH1λ+
genomic DNA titrated from 105 to 10 copies.
See application note 63-0054-46 for comparison details.
Plasmid DNA template was amplified with the TempliPhi 100
Amplification Kit and subsequently sequenced using
DYEnamic™ Terminator Cycle Sequencing Kit and analyzed
on an ABI PRISM™ 3100 Genetic Analyzer.
Cloning and
Sequencing
DNA clean-up:
• illustra GFX™ PCR DNA and Gel Band Purification Kit:
This kit is designed for the fast isolation and concentration
of high-quality DNA fragments from PCR mixtures, DNA
containing agarose gel bands, enzyme-based DNA modifications, and restriction digestions. You can obtain high-quality
DNA in a flexible 10 to 50 μl elution volume for downstream
cloning applications with a simplified single kit design
A
• illustra AutoSeq™ G-50: for rapid removal of unincorporated
fluorescent dye-terminators from automated sequencing
reactions, ready-to-use format allows less than 4 min
process time
Gene Expression
Sample preparation
for gene expression
RNA sample preparation
RT-PCR
Obtaining high-quality total RNA is important for gene
expression analysis methods such as microarray, real
time PCR and Northern blot studies. illustra products
offer several high-quality, easy-to-use kits for total mRNA
purification and RT-PCR assays.
illustra RTG RT-PCR Beads: one-tube, one-step RT-PCR
reaction in preformulated, predispensed, room temperature
stable bead format, ensure greater reproducibility between
reactions, minimize pipetting steps and reduce the potential
for pipetting errors and contamination.
• illustra RNAspin Mini Isolation Kit: for rapid high-quality
total RNA isolation from diverse sample types; DNase I
included for the removal of genomic DNA
B
• illustra RNAspin Midi Isolation Kit: for large-scale highquality total RNA isolation from diverse sample types;
DNase I included for the removal of genomic DNA
RT-PCR
product
primer
High-quality total RNA is generated time after time
105
104
103
330
100
33
10
3
0
Number of HeLa Cells input into RNAspin Mini
illustra RNA isolation and RT-PCR protocols work great
together, shown by the RT-PCR of total RNA isolated with
the RNAspin Mini Isolation Kit standard protocol from 105,
104, 103, 330, 100, 33, 10, 3 and 0 HeLa cells. The RT-PCR
was performed using 3 μl of total RNA (each elution volume
40μl). The specific RT-PCR product, generated from β-actin
primers, is 626 bp in length.
illustra GFX PCR DNA and Gel Band Purification Kit performs
functionally equivalent to the four Qiagen kits (QIAquick™
and MinElute™ PCR Purification Kits, and QIAquick and
MinElute Gel Extraction Kits) required to obtain the same
results. DNA purified from (A) PCR mixtures and (B) DNA
containing agarose gel bands was used in cloning experiments.
White colonies indicate successful cloning. See data file
28-9075-94 for comparison details.
The RNAspin Midi kits produce high-quality RNA: rRNA bands
that are sharp, with the 28S band being about twice as intense
as the 18S band, and with good RIN values. Total RNA from
five independent 100 mg rat liver samples was isolated with
RNAspin Midi and evaluated using the Agilent 2100 bioanalyzer.
Genotyping
Sample preparation
for genotyping
Genomic DNA preparation
1
2
3
QIAamp
4
5
1
2
3
4
M
5
• illustra tissue and cells genomicPrep Mini Spin Kit: rapid
small-scale isolation of high molecular weight genomic
DNA from a variety of animal tissues and mammalian cell
cultures; reduces total preparation time by 90 min
• illustra blood genomicPrep Mini Spin Kit: rapid, small-scale
isolation of genomic DNA in less than 20 min from a wide
range of blood sample types with increased purity and yield
• illustra GenomiPhi V2 DNA Amplification Kit: simplified
small-scale genomic DNA preparation through whole
genome amplification, enabling DNA preparation from
small quantity source materials for cloning, array CGH,
SNP and STR genotyping
1. Mouse (Mu)-Bone Marrow
2. Mu-Blood K3EDTA
3. Human (Hu)-K3EDTA
4. Hu-Citrate
5. Hu-Heparin
M = HindIII Marker
Specification for illustra tissue and cells genomicPrep Mini Spin Kit.
illustra tissue and cells genomicPrep Mini Spin Kit is designed
for the rapid extraction and purification of high molecular
weight genomic DNA from a variety of animal tissues and
mammalian cell cultures. Optimized lysis and protocol cut
total preparation time to just 90 min for tissues and 45 min
for cells (from sample to elution).
Feature
High-quality genomic DNA preparation from blood
illustra blood genomicPrep Mini Spin Kit is designed for the
rapid extraction and purification of high molecular weight
genomic DNA from whole blood, buffy coat, bone marrow,
and nucleated red blood cells. The procedure is rapid, but
with reduced shearing, over 90% of resulting genomic DNA
is intact with averages sizes > 20 kb.
illustra blood genomicPrep Mini Spin Kit gives better yield
and purity compared to QIAamp Blood Mini Kit in the
presence of three commonly used anticoagulants: EDTA,
Citrate, and Heparin. Manufacturers’ recommended
methods were followed.
illustra tissue and cells genomicPrep Mini Spin Kit
10
Up to 50% reduction in protocol time for small-scale genomic
DNA preparation from tissues and cells
Genotyping
Good quality genomic DNA is critical to the success of any
investigation into genetic diseases. Obtaining a large quantity
of high-quality genomic DNA for genotyping research is often
laborious and tedious. illustra genomic DNA preparation
products and downstream PCR solutions produce exceptional
genomic DNA yields and quality for genotyping.
illustra
Specification
Sample type
Animal tissue
Cultured cells
Time/prep
90 min
45 min
Sample input size
5 to 50 mg of animal tissue
Up to 5 x 106 cultured cells
Elution volume
200 µg
200 µg
Number of steps
5
5
Binding capacity
> 35 µg
> 35 µg
Typical yield
0.5 –1.5 µg DNA/mg 10 to 20 µg of gDNA of animal tissue1.
(from 5 x 106 cells)
Purity (A260 /A280)
> 1.75
> 1.75
Product size
> 20 kb
> 20 kb
1. Values shown from rat liver samples; yields will vary depending in tissue
type used.
Yields and purities of genomic DNA isolated from the blood of
various species with the illustra blood genomicPrep Mini Spin Kit
and the QIAamp Blood Mini Kit from Qiagen. Samples = 200 μl
K3-EDTA whole blood, n = 3.
11
Genotyping
Representative whole-genome amplification of genomic DNA
PCR
DNA clean-up
illustra GenomiPhi V2 DNA Amplification Kit is designed
for the quick preparation of 4 to 7 μg of genomic DNA
from different types of samples such as whole blood, FTA
card, buccal swab, microbial cells, animal tissues, etc.
The amplified DNA is representative and of a sufficient
quality to be used directly for downstream PCR, genotyping
and cloning applications.
We offer a broad portfolio of high-fidelity PCR enzymes
to meet stringent demands of high accuracy and yields.
• illustra GFX PCR DNA and Gel Band Purification Kit:
one kit does it all – purify PCR products from reactions
or agarose gel bands: offering maximum flexibility with
elution volumes from 10 to 50 µl
• MicroSpin™ G-25: For rapid buffer exchange/desalting
of PCR products and other DNA samples in a volume
of 10 to 100 μl using spin-column chromatography
Genotyping
• illustra Hot Start Mix RTG: room temperature stable,
premixed, predispensed reactions for Hot Start PCR
featuring high-performance PuReTaq DNA polymerase
Hot Start PCR bead improves PCR specificity
Individual human genomic DNA (gDNA) obtained from Coriell
was amplified with GenomiPhi kits and subjected to analysis
on Affymetrix™ 10K SNP chip (Green = % call rate, Blue = %
concordance).
Amplification yields for GenomiPhi V2 DNA Amplification Kit
using purified DNA (10 ng) or nonpurified cell lysates.
illustra Hot Start Mix RTG is a premixed, predispensed,
ambient temperature-stable formulation. It is based
on a novel PCR method that uses a hot start activator
protein to sequester primers prior to PCR. It effectively
reduces nonspecific priming and primer-dimer formation
during PCR.
1
2
3
4
5
6
illustra Hot Start Mix RTG
illustra Hot Start Mix RTG gives exceptional amplification
performance compared to Taq DNA polymerase, as
illustrated by this amplification of a 1018-bp fragment
of a human gene. In all the reactions, 2 ng of human
genomic DNA and 10 pmol each of forward and reverse
primers specific for the 1018-bp fragment were used.
Lanes 1 and 2 contain Hot Start Mix RTG, and lanes 4 and
5 contain AmpliTaq DNA Polymerase. No-template control
reactions for each sample are in lanes 3 and 6.
GenomiPhi V2 DNA Amplification Kit
12
13
Precious protein samples
deserve the best possible start
Proteins
Results from protein analyses can only be as good as the
initial samples you start with. To help you achieve great
results, we have designed Trap products specifically for
the intended application:
Reproducibility
Yield
Purity
Designed for uncompromising reproducibility, Trap products
provide reliable performance run after run. 96 desalting runs
on the PD MultiTrap™ G-25 96-well filter plate showed 93%
desalting capacity and well-to-well variation of 1%.
With difficult and precious samples, optimizing the yield of
protein is critical. All Trap formats are developed to maximize
yield. Protein enrichment with Protein A SpinTrap™ showed
four-times higher recovery of target proteins compared with
Pierce Seize™ Classic (A).
After reproducibility and yield, purity is critical. High purity
eliminates complications in downstream work and
minimizes further handling steps. In the example below,
Protein G HP SpinTrap enriched the target protein and
removed contaminants, with the major amount of the
protein recovered in the first elution fraction.
• Trap product protocols focus on preparing proteins for
downstream analysis methods such as electrophoresis,
liquid chromatography and mass spectrometry
• We package the products in appropriate formats for
typical sample volumes and numbers for small series
as well as for high-throughput requirements
Start material
First elution fraction
Second elution fraction
Third elution fraction
Proteins
• Our application-driven concept makes it easier to find
the right sample prep product and assures you that all
the requirements are met for typical analysis situations
• Trap provides Optimization Guides to tailor the protocol
to your protein
All Trap products are engineered to provide uncompromising
levels of reproducibility, yield and purity for protein sample
preparation.
Removal of NaCl from BSA on a PD MultiTrap G-25 96-well
filter plate showed highly reproducible results. The average
desalting capacity was 93% and the well-to-well variation
was 1% RSD.
Protein A SpinTrap delivers four-times higher recovery as
compared with Pierce Seize Classic (A). In this example,
the enrichment of Cy™5 labeled transferrin from an E. coli
lysate was compared. See data file 28-9067-89 AB for
comparison details.
Enrichment of Cy5-labeled human serum transferrin from
an E. coli lysate using α-transferrin antibodies cross-linked
to Protein G SpinTrap. Elution fractions were analyzed by
SDS-PAGE. The gel was stained using Deep Purple™ Total
Protein Stain (green) and Cy-5 labeled transferrin (red).
SpinTrap
MultiTrap
14
GraviTrap™
15
Sample preparation
for electrophoresis
2-D Clean-Up Kit
Clean-up
Appropriate sample preparation is absolutely essential for
achieving good electrophoresis results. Treatments that
optimize the solubilization of the different proteins and
remove contaminants in the sample are vital, otherwise
streaking or incompletely focused spots may occur.
Elimination of interfering substances in 2-D electrophoresis
The 2-D Clean-Up Kit prepares samples for 2-D electrophoresis
that might otherwise produce poor 2-D spot-maps due to high
conductivity, high levels of interfering substances, or low
protein concentration. The reagents precipitate proteins
quantitatively while leaving interfering substances such as
detergents, salts, lipids, phenolics and nucleic acids in solution.
Protease Inhibitor Mix, Nuclease Mix
Enzyme regulation
Proteases may be released upon cell disruption. Proteolysis
greatly complicates analysis of the 2-D result, since multiple
spots occur from the degradation products. Protein samples
should be protected from proteolysis during cell disruption
and subsequent preparation.
Prevent proteolysis and remove undesired nucleic acids
in cell lysis
Sample preparation often includes the inhibition of protease
activity. This requires the use of an optimized concentration
of reversible and irreversible inhibitors for optimal action.
The Protease Inhibitor Mix offers this combination with
excellent inhibition of protease activities and the resultant
protection of proteins during purification from animal tissues,
plant tissues, yeast and bacteria.
Electrophoresis
Removal of nucleic acids is often required to avoid contamination and subsequent artifacts on electrophoresis gels.
Nuclease Mix offers an effective mix of enzymes with DNase
and RNase activities, as well as the necessary co-factors for
optimal nuclease activity.
Electrophoresis
Horizontal streaking caused by residual SDS is eliminated.
Outer membrane protein F-precursor is normally difficult to
isolate from 2-D gels due to low abundance, but is successfully identified from a 2-D gel using 2-D Clean-Up Kit for
sample clean-up prior to 2-D DIGE analysis. In this example
the method also allowed identification of two isomers of
the protein (peaks a and b), which were not observed in the
untreated sample.
The Protease Inhibitor cocktail effectively
inhibits over 90% of protease activity.
16
The Nuclease Mix offers an effective mix of
DNase and RNase enzymes and the necessary
co-factors for optimal nuclease activity.
Compatible with Protease Inhibitor.
17
Sample preparation for
liquid chromatography
Flexible protocols for optimal results
PD-10 Desalting Columns, PD MidiTrap G-25, PD MiniTrap
G-25, PD SpinTrap G-25, and PD MultiTrap G-25
Gel filtration, based on Sephadex™ G-25 media, allows group
separation of biomolecules with a molecular weight above
5000 from contaminants such as salts, dyes, and radioactive
labels. By using a centrifuge-based protocol, less dilution
of the eluted samples occurs in comparison to gravity-flow
based methods.
Desalting and buffer exchange/clean-up
For applications such as liquid chromatography, the buffer
composition of the protein sample is vitally important.
Proteins can be reproducibly transferred from one buffer
to another using a method called buffer exchange. PD-10
Desalting Columns, PD MidiTrap™ G-25, PD MiniTrap™G-25,
PD SpinTrap G-25 and PD MultiTrap G-25 are prepacked
single-use columns or 96-well filter plates developed for
buffer exchange or clean-up of biological samples.
The gravity protocol, however, provides the highest desalting
capacity and recovery (see figures below). The aims of the
preparative step determines the choice of protocol. If avoiding
dilution of the sample is critical, choose the centrifugation
protocol; if the recovery of the protein is critical, the gravity
protocol is preferable.
Sample volume
(spin)
PD MultiTrap G-25
–
70–130 μl
PD SpinTrap G-25
–
70–130 μl
PD MiniTrap G-25
0.1–0.5 ml
0.2–0.5 ml
PD MidiTrap G-25
0.5–1.0 ml
0.75–1.0 ml
PD-10 Desalting Columns
1.0–2.5 ml
1.75–2.5 ml
The PD line of columns and plates covers the sample volume
range from 70 μl to 2.5 ml.
Trap prepacked PD products provide:
• Convenient and rapid clean-up with high reproducibility
Liquid
Chromatography
Sample volume
(gravity)
• A wide range of applications such as desalting,
buffer exchange and removal of low-molecular
weight compounds
• High desalting capacity
Liquid
Chromatography
• The versatility to also use a centrifugation protocol
with all gravity columns
Simple clean-up and removal of NaCl from BSA using one or
several columns in parallel with gravity flow, without the need
for a purification system. The sample was 1000 μg/ml bovine
serum albumin (BSA) in 1 M NaCl and the PD MiniTrap G-25
was used. The protein recovery was 95% and the desalting
capacity was 99%.
The PD-10 family of desalting and buffer exchange columns.
18
19
Sample preparation
for mass spectrometry
Protein G HP MultiTrap, Protein G HP SpinTrap columns,
Protein G HP Sepharose, Phos SpinTrap Fe
Parallel affinity-based sample enrichment improves
MS results
High confidence in protein identification is assured
after enrichment
Protein enrichment by immunoprecipitation
Robust and reproducible results were achieved with all formats,
however the Protein G HP MultiTrap yields superior results for
average recovery.
Comparing LC-MS/MS protein identifications of starting material
and the eluate after immunoprecipitation reveals that the
sequence coverage is highly improved after enrichment.
Serotransferrin was not found in the starting material, but
was the highest ranked protein in the enriched sample, with
a sequence coverage of 70%.
Enrichment with Phos SpinTrap Fe gives high purity enrichment
of phosphorylated peptides with excellent reproducibility.
A
A
2900
2700
2500
2300
A
300
1100
1300
1500
2066.952
2088.958
1880.875
1724.816
1700
1951.793
700
500
Microcentrifuge tube
1676.817
900
1746.786
1501.805
1439.798
1500
1523.782
1900
1700
1589.706
2100
1300
1100
1900
2100
2300
2500
2300
2500
m/z
2061.819
2900
2700
2500
2300
*
*
Mass
Spectrometry
Protein G HP MultiTrap yields superior results for average
recovery after immunoprecipitation
B
B
B
2100
1900
1700
1500
1992.207
Intensity
1300
1100
20
* Serotransferrin precursor
Serotransferrin was not found in (A) the starting material, but
was the highest ranked protein in (B) the enriched sample,
marked with an asterix. The graphs show proteins identified
(x-axis) vs. sequence coverage (y-axis).
900
700
500
300
1100
1300
1500
2005.073
The experimental workflow shows the immunoprecipitation
of human serotransferrin (hTf) using rabbit polyclonal
antibodies in 96-well plates, spin columns and batchwise
in microcentrifuge tubes. The original hTf concentration
was equal to 0.15% specific purity.
1479.875
Mass
Spectrometry
2044.987
1479.796
The enrichment resulted in a 100-fold increase from 0.15%
to about 15% purity for serotransferrin.
Immunoprecipitation enrichment methods are compatible
with down­stream LC-MS/MS analysis. Enrichment vastly
improves protein purity and gives better sequence coverage
and identification results. Robust, reproducible and high-yield
results can be obtained by using Protein G HP MultiTrap plates.
Phos SpinTrap Fe is designed for the capture and enrichment
of microgram quantities of phosphopeptides from protein
digests of cell culture lysates, as well as body fluids and tissues
prior to mass spectrometry analysis. For simplification, the
protocol is optimized with elution conditions to fit specific
downstream analyses such as mass spectrometry.
1567.745
Enrichment improves purity 100-fold
In the subsequent LC-MS/MS analysis, around 50 proteins
were identified in the different samples. The proteins found in
the starting material, however, were mainly highly abundant
E. coli proteins, which could only be identified by one or two
peptide fragments.
Phosphoproteins are frequently expressed at very low concentrations and ionize poorly, making their detection by MS difficult.
Intensity
Protein separation and analysis is challenging due to
the complexity and dynamic range of different proteins.
Enrichment is often desired to increase the signal in
subsequent analysis (e.g. MS). Robust and reproducible
methods are therefore needed for the preparation of
samples. In this example, different formats of Protein G
Sepharose™ High Performance were compared in an
immunprecipitation protocol with the immobilization of
an antibody from a clarified cell lysate. The antibody
coupled to Protein G Sepharose High Performance was
used for capture and enrichment of human transferrin
in an E. coli extract. Recovery using different formats was
compared including Protein G HP MultiTrap, (96-well filter
plates), Protein G HP SpinTrap columns, and batch-wise
adsorption using bulk medium in microcentrifuge tubes.
Outstanding enrichment of phosphorylated peptides
1700
1900
2100
m/z
MALDI-TOF spectra of (A) starting material and (B) enriched
material eluted from Phos SpinTrap Fe column. The enriched
monophosphopeptide is marked with an asterisk.
21
Sample preparation for
tagged protein expression
HisTrap, His SpinTrap, His MultiTrap, His GraviTrap, GSTrap, GST SpinTrap Purification Module, GST MultiTrap
Recombinant protein capture
The development of protocols for efficient approaches
to cloning, expression, purification and crystallization
of a large numbers of tagged recombinant proteins is
essential to shorten the time from gene to drug target.
A parallel setup can increase throughput in each step.
Prepacked formats give increased accuracy and robustness of the analysis.
• Products prepacked with Ni Sepharose media for the
purification of histidine-tagged proteins are characterized by low nickel leakage and are compatible with
a wide range of additives used in protein purification.
Moreover the binding capacity is high, which gives lower
costs per prep
• Products packed with Glutathione Sepharose media for
purification of GST-tagged proteins offer mild, nondenaturing conditions that preserve protein antigenicity and
function. In addition, high purity is achieved from a single
purification step
Maltose binding protein tag (MBP)
Streptavidin tag
Tagging proteins with MBP often gives increased expression
levels and higher solubility of the target protein. Since
MBP increases solubility, the tag is particularly useful for
recombinant proteins accumulated in an insoluble form
(inclusion bodies).
Strep(III) tag is a small tag of only eight amino acids. Due to the
small size of the tag, it does not need to be removed before
performing structural and functional studies. The Strep(III) tag
binds specifically to the StrepTactin ligand enabling easy
purification.
MBPTrap™ HP is a ready-to-use HiTrap column for purification
of recombinant proteins tagged with maltose binding
protein (MBP).
StrepTrap™ HP is a ready-to-use HiTrap column for purification
of Strep(II)-tagged recombinant proteins with a high degree of
purity and convenience.
Recombinant proteins with histidine or GST tag
Easy scale-up with reliable results
Highly pure MBP-tagged recombinant proteins can be eluted
in concentrated form and small volumes:
Highly pure Strep(II)-tagged recombinant proteins can be
eluted in concentrated form and small volumes:
Screening experiments and small-scale or highthroughput parallel purifications of histidine- and
GST-tagged proteins are easily performed using
GraviTrap™, SpinTrap or MultiTrap formats.
• For automated and manual screening of large sample
series, use the MultiTrap format
• Physiological conditions and mild elution preserve target
protein activity
• Physiological conditions and mild elution preserve target
protein activity
• For rapid screening of a smaller sample series or single
samples, use the SpinTrap format
• Compatible with commonly used aqueous buffers and
easily regenerated using 0.5 M NaOH
• Fast and easy regeneration with 0.5 M NaOH
• Reproducible: less sample pretreatment is needed;
loading unclarified sample directly increases
reproducibility in results, and saves time
• For running larger sample volumes for reproducible
manual preparations, select the GraviTrap format
• Ease of use: simple handling and consistent results
All these formats can easily be scaled up to the HiTrap™
format for use on ÄKTAdesign™ purification systems.
As the same conditions (e.g. buffers, concentrations,
gradients, etc.) apply at all scales, scaled-up purification
results are consistent and optimization times are short.
• Compatible with a wide range of reducing agents
Expression Tag
His SpinTrap works with unclarified samples,
saving time and avoiding loss of material
from the centrifugation step.
22
Expression Tag
Purification of unclarified samples in 10 min
His SpinTrap columns enable capture of histidine-tagged
proteins directly from unclarified cell lysates. Purifying
unclarified samples reduces prep time by as much as one
hour by eliminating the clarification step, which consequently reduces the risk of losing target protein. The short
preparation time minimizes degradation and oxidation of
sensitive target proteins.
MBPTrap HP, and StrepTrap HP 1ml and 5ml
23
Sample preparation for
protein expression
Ab SpinTrap
One-step antibody purification
The high specificity of Protein A and Protein G for the Fc
region of antibodies provides a fast, reliable approach for
achieving efficient purification of antibodies. By coupling
Protein A or Protein G to Sepharose High Performance,
both high selectivity and resilience to a range of buffer
conditions are enabled, thus supporting a wide range of
purification conditions.
These products provide easy scale-up, both with regard
to amount of protein to be prepared and number of
samples that can be processed, with reproducible results.
The smaller-scale products in the form of Ab SpinTrap,
Protein G HP SpinTrap and Protein A HP SpinTrap are
suitable for rapid screening of a number of different
clones. At a later stage these can be easily scaled up
using the same media in HiTrap format for larger scale
production. If a larger number of clones in small scale
need to be processed, the Protein G HP MultiTrap and
Protein A HP MultiTrap are suitable in automated settings.
Polyclonal antibodies are commonly used as reagents
in immunochemical methods, using crude serum as the
source. High purity of antibodies from serum can be
achieved (> 95% purity) in one step in less than 20 min.
90% sample purity in 20 min
Purification of antibodies from serum without centrifugation,
dilution or filtration is made possible by Ab SpinTrap columns.
Binding, elution and neutralizing buffers are easily prepared
using the buffer concentrates provided in the Ab Buffer Kit.
In this example, the undiluted serum from an immunized rabbit
was purified using Ab SpinTrap. Purity, as measured by SDSPAGE analysis, exceeded 90%, and the yield, as determined
by A280, was 2 mg.
For antibody purification, the Trap platform provides:
• Convenient and quick purification due to the prepacked
format and absence of sample pretreatment
Purification of antibodies from unclarified serum
using Ab SpinTrap.
• High purity and high yield of antibodies
SDS-PAGE
Lane
1. Low molecular weight markers
2. Eluted pool (diluted 1:5)
3. Start material (diluted 1:50)
Expression
• Simple and robust methods for reproducible results
Expression
Ab SpinTrap and Ab Buffer Kit
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Ab SpinTrap
25
Get it right from the start
No matter what type of research you are conducting, the results will
only be as good as your initial sample preparation. The illustra and Trap
platforms provide uncompromising standards of quality for protein and
nucleic acid preparation. They are flexible, easy to use products that
provide rapid protein, DNA and RNA sample preparation, and enable
you to get the best possible start to your research.
GE Healthcare’s comprehensive sample preparation offering can easily
be ordered online at:
www.gelifesciences.com/sampleprep
“The illustra plasmidPrep mini Kit is a major innovation. With a culture
volume of 3 ml, we were able to quickly produce 25 µg of pure plasmid
vector DNA that could easily be reproduced. And after concentrating
the sample to 1 µg/µl with a SpeedVac™ for 10 min, we obtained the
same high transfection rate with Mycoplasma cells that we used to get
with the Qiagen Midi kit. A great material and time saver .”
Michaela Rode, EMBL, Germany
“Protein Sample preparation is often considered less important than other
major steps in protein research, but the success of a project frequently
depends on the quality of the starting material. Far greater attention to
sample preparation is needed due to the nature of biological samples
and the complexity of proteins in cells. Sample preparation is critical
to acquiring better data and thus achieving more complete conclusions
from experimental work.”
Professor Mathias Uhlén, Royal Institute of Technology, Stockholm
“The beginning is the most important part of any work.”
Plato’s Republic, Book II
27
Testimonial
26
GE, imagination at work, and GE monogram are
trademarks of General Electric Company.
AutoSeq, ÄKTAdesign, ÄKTAxpress, Cy, Deep Purple,
DYEnamic, Ettan, GenomiPhi, GFX, GraviTrap, HiLoad,
HiPrep, HisTrap, HiTrap, illustra, MicroSpin, MidiTrap,
MiniTrap, MBPTrap, MultiTrap, PuReTaq, Ready-To-Go,
SpinTrap, StepTrap, Sephadex, Sepharose, Superdex, and
TempliPhi are trademarks of GE Healthcare companies.
This product is covered by US pat No 6 623 655 and
their equivalents in other countries. Purification and
preparation of fusion proteins and affinity peptides
comprising at least two adjacent histidine residues
may require a license under US pat 5,284,933 and US
pat 5,310,663, including corresponding foreign patents
(assignee: Hoffman La Roche, Inc).
GST Gene Fusion Vectors: A license for commercial use
of GST Gene Fusion Vectors under US patent 5,654,176
and equivalent patents in other countries must be
obtained from Millipore Corp (formerly Chemicon
International Inc).
IMAC Sepharose products, Ni Sepharose products
and Fe Sepharose products are covered by US pat
No 6 623 655 and its equivalents in other countries.
Phi 29 DNA Polymerase: Phi 29 DNA polymerase and
its use for DNA synthesis is covered by US patent
numbers 5,854,033, 5,198,543, 5,576,204 and 5,001,050.
StrepTrap HP: StrepTrap HP has been manufactured
by GE Healthcare and contains Strep-Tactin, manufactured by IBA GmbH, which has been immobilized to
GE Healthcare’s chromatography media. Strep-Tactin is
covered by US patent number 6,103,493 and equivalent
patents and patent applications in other countries.
The purchase of StrepTrap HP includes a license under
such patents limited to internal use, but not re-sale.
Please contact IBA for further information on licenses
for commercial use of Strep-Tactin.
Taq DNA Polymerase: Use of this product is covered
by one or more of the following US patents and corresponding patent claims outside the US: 5,079,352,
5,789,224, 5,618,711, 6,127,155, 5,407,800, 5,322,770,
5,310,652, and claims outside the US corresponding to
US Patent No. 4,889,818. The purchase of this product
includes a limited, non-transferable immunity from suit
under the foregoing patent claims for using only this
amount of product for the purchaser’s own internal
research. No right under any other patent (such as the
patented 5’ Nuclease Process claims in US Patents
Nos. 5,210,015 and 5,487,972) and no right to perform
commercial services of any kind, including without
limitation reporting the results of purchaser’s activities
for a fee or other commercial consideration, is conveyed
expressly, by implication, or by estoppel. This product
is for research use only. Diagnostic uses under Roche
patents require a separate license from Roche. Further
information on purchasing licenses may be obtained by
contacting the Director of Licensing, Applied Biosystems,
850 Lincoln Centre Drive, Foster City, California 94404, USA.
GE Healthcare UK Limited
Amersham Place
Little Chalfont
Buckinghamshire, HP7 9NA
UK
GE Healthcare Europe, GmbH
Munzinger Strasse 5
D-79111 Freiburg
Germany
GE Healthcare Bio-Sciences Corp.
800 Centennial Avenue, P.O. Box 1327
Piscataway, NJ 08855-1327
USA
GE Healthcare Bio-Sciences KK
Sanken Bldg., 3-25-1, Hyakunincho
Shinjuku-ku, Tokyo 169-0073
Japan
ABI PRISM is a trademark of Applera Corp. AmpliTaq
and AmpliTaq Gold are trademarks of Roche Molecular
Systems Inc. QIAamp, Qiagen, and QIAprep are trademarks of the Qiagen Group. Seize and SpeedVac are
trademarks of Thermo Fisher Scientific Inc.
© 2008 General Electric Company—All rights reserved.
First published Dec. 2007
All goods and services are sold subject to the terms and
conditions of sale of the company within GE Healthcare
which supplies them. A copy of these terms and
conditions is available on request. Contact your local
GE Healthcare representative for the most current
information.
For contact information for your local office,
please visit: www.gelifesciences.com/contact
GE Healthcare Bio-Sciences AB
Björkgatan 30
751 84 Uppsala
Sweden
www.gelifesciences.com/sampleprep
28-9320-93 AB 04/2008
28