5.
Protein and Organelle Isolation
Improved Protein Research with Dynabeads®
59
•
59
Advantages of Dynabeads® Magnetic Protein Separation
Choosing the Right Product
•
A Wide Variety of Available Dynabeads®
6o
Magnetic Isolation of Proteins with Dynabeads®
•
Choosing the Size
•
Hydrophobic or Hydrophilic?
62
Examples of Protein Applications
•
Immunoprecipitation and Co-Immunoprecipitation
•
“Pull-Down” of Large Protein Complexes
•
Protein Isolation Through Tags
•
Immunoassays
•
Phage Display Panning
•
Recombinant Protein Isolation
•
Downstream Mass Spectrometry Analysis
•
Intellectual Property
63
Automation
65
Subcellular Fractionation with Dynabeads®
•
The Product of Choice for Subcellular Fractionation
•
Overcoming the Limitations of Other Techniques
•
Application Guide – Subcellular Fractionation
66
Visit www.dynalbiotech.com for the latest product up-dates and references.
www.dynalbiotech.com
57
60
62
62
63
63
64
64
64
65
65
65
66
67
67
Application Overview
Samples:
Whole Blood
Cord Blood
Mononuclear Cells
Bone Marrow
Buffy Coat
Spleen
Lymph Node
Tissue Digest
Depletion
Positive Isolation
Expansion
Negative Isolation
DNA Isolation
Organelle Isolation
mRNA Isolation
Specific Protein Isolation
Capture of Biotinylated Target
58
www.dynalbiotech.com
Improved Protein Research
Protein and Organelle Isolation
Improved Protein Research with Dynabeads®
Magnetic separation provides the opportunity to work with concentrated
protein solutions throughout the isolation procedure, preserving large protein
complexes and the proteins’ native state. Magnetic solid-phase technology
can be used in protein isolations, protein pull-down, immunoprecipitations,
bio-assays and bio-panning. Magnetic isolation based on protein-protein
interaction has become an efficient and gentle tool, alone or in combination
with other techniques, in the growing field of proteomics.
All Dynabeads® methods are gentle to your target and offer benefits in terms
of reproducibility and sensitivity. The monodisperse shape and smooth
surface of the Dynabeads® (fig. 1) allow for optimal accessibility of proteins
to the surface and optimal binding kinetics. Their defined iron content also
ensures a strong and even attraction to the magnet.
Fig. 1: Dynabeads® are the only uniform magnetic
beads available. The monodispersity of the beads
offers benefits extending far beyond their unique
visual appearance, ensuring maximum efficacy
and reproducibility.
5
Enzymatic reactions are not inhibited by Dynabeads®, and bead-bound
material can be included in downstream magnetic handling as well as
detection assays.
Protein and Organelle Isolation
All Dynabeads® protocols can easily be automated on liquid handling
platforms, due to the truly uniform characteristics of the Dynabeads® (see fig.
1 and 2) as well as their excellent binding kinetics. No magnetic remanence
when removed from a magnetic field combined with good dispersion abilities,
allow for simple and effective washing. For high-throughput and large scale
applications with automation, please contact Dynal Biotech for additional
information.
Advantages of Dynabeads® Magnetic Protein Separation:
• Effective and rapid solid-phase isolation based on excellent liquid phase
kinetics.
• Magnetic separation allows easy washing and concentration of your
target.
Flexible all-in-one-tube isolation, avoiding columns and centrifugations.
Efficient and cost-effective isolation from small volumes.
Minimal loss and high reproducibility enable quantitative isolations.
Simple handling of superparamagnetic beads facilitates automation.
Low non-specific binding due to optimal surface chemistries.
Iron is sealed inside the beads and bound molecules are not exposed to
iron.
• Gentle isolation by binding based only on affinity interactions.
•
•
•
•
•
•
www.dynalbiotech.com
59
Fig. 2: Size distribution of Dynabeads® MyOne™,
showing their true monosized characteristics.
A l l D y n a b e a d s ® h a ve b e e n t h o r o u g hl y
characterised and are manufactured with
highly controllable product properties and a
unique level of reproducibility both within and
between batches.
Choosing the Right Product
Product
Characteristics
Dynabeads® Protein A
2.8 µm beads
Fc-binding.
with covalently bound, high Up to 250 µg human
quality protein A.
IgG or 100 µg mouse
IgG per ml.
Human IgG1, 2, 4 - Mouse IgG2a, 2b, 3 10 min.
- Rat IgG2c - Bovine IgG2 – Canine IgG - at room
Goat IgG2 - Guinea Pig IgG - Monkey IgG temperature.
- Porcine IgG - Rabbit IgG – Sheep IgG2.
Dynabeads® Protein G
2.8 µm beads
Fc-binding.
with covalently bound, high Up to 250 µg human
quality protein G.
IgG or 640 µg mouse
IgG per ml.
Human IgG1, 2, 3, 4 – Mouse IgG1, 2a,
2b, 3 - Rat IgG2a, 2c – Bovine IgG1, 2
- Goat IgG1, 2 – Guinea Pig IgG - Horse
IgG - Monkey IgG – Porcine IgG - Rabbit
IgG - Sheep IgG1, 2.
10-40 min.
at room
temperature.
Dynabeads® M-280
Sheep anti-Mouse IgG
2.8 µm beads
with covalently bound,
affinity purified polyclonal
sheep anti-mouse IgG
antibodies.
Binds randomly, but
mostly Fc binding.
Mouse IgG1, IgG2a, IgG2b.
> 30 min.
at 2-8o C.
Dynabeads® M-280
Sheep anti-Rabbit IgG
2.8 µm beads
with covalently bound,
affinity purified polyclonal
sheep anti-rabbit IgG
antibodies.
Binds randomly, but
mostly Fc-binding.
Any Rabbit IgG antibody.
> 120 min.
at 2-8o C.
Dynabeads® M-280
Streptavidin
2.8 µm hydrophobic beads
with covalently bound
streptavidin.
Binds up to 5-10 µg
Any biotinylated antibody, as well as
biotinylated antibodies other biotinylated ligands.
per mg.
30 min.
at room
temperature.
Dynabeads® MyOneTM
Streptavidin C1
1.0 µm hydrophilic beads
with covalently bound
streptavidin.
Binds up to 15-20 µg
Any biotinylated antibody, as well as
biotinylated antibodies other biotinylated ligands.
per mg.
30 min.
at room
temperature.
Dynabeads® MyOneTM
Streptavidin T1
1.0 µm hydrophobic beads
with covalently bound
streptavidin.
Binds up to 10-25 µg
Any biotinylated antibody, as well as
biotinylated antibodies other biotinylated ligands.
per mg.
30 min.
at room
temperature.
1.0 µm beads with BD
TALONTM chemistry.
Binds approximately
10 µg tagged proteins
per mg beads.
10 min.
at room
temperature.
New
Binding Capacity
Type of Ligand
Coupling
uct
prod
Dynabeads® TALONTM*
Any histidine affinity-tagged proteins
with non-adjacent histidine residues.
Table 1. Product Guide - Pre-coated Dynabeads®
* The IMAC technology for Dynabeads® TALON™ is licensed from BD Biosciences Clontech.
Choosing the Right Product
A Wide Variety of Available Dynabeads®
Magnetic handling is very simple with few manipulations required. As
described in chapter 2, there are a variety of method options for sample
preparation, handling and analysis. The method of choice will depend upon
the type of target and your specific application. Tables 1 and 2 describe the
main features of the different Dynabeads® available for protein research.
Dynabeads®-based technology utilises the affinity interactions between
bead-bound ligands and their specific targets. The available Dynabeads®
products fall into two categories:
60
www.dynalbiotech.com
Choosing the Right Product
Product
Characteristics Binding Chemistry Binding Properties
Dynabeads®
1.0 µm hydroMyOneTM Tosylactivated phobic beads
with surface
tosyl groups.
Covalent binding to
primary amino and
sulphydryl functional
groups.
Dynabeads®
1.0 µm hydroMyOneTM Carboxylic Acid philic beads
with surface
carboxylic acid
groups.
Covalent amide bonds ·
with primary amino
groups in proteins and ·
peptides.
·
Dynabeads®
M-280 Tosylactivated
2.8 µm hydrophobic beads
with surface
tosyl groups.
Covalent binding to
primary amino and
sulphydryl functional
groups.
·
·
·
2.8 µm hydrophilic beads
with surface
epoxy groups.
Covalent binding to
primary amino and
sulphydryl functional
groups.
2.8 µm hydrophilic beads
with surface
amino groups.
Covalent binding
through reductive
amination of
aldehydes or
activated carboxylic
acids.
O
OS
·
·
·
Coupling
No further activation required.
Binds via the antibodies’ Fc part.
Binds any antibody or protein
ligand.
Overnight coupling at
neutral to high pH and high
temperature.
100% covalent binding, activation
through carbodiimide is required.
Random binding of antibodies and
N-terminal coupling of peptides.
Low non-specific binding of nucleic
acids.
No further activation required.
Binds via the antibodies’ Fc part.
Binds any antibody or protein
ligand.
Immediate peptide bond
formation at pH 5 and
room temperature.
·
·
·
No further activation required.
Random binding of antibodies.
Binds functional enzymes.
Overnight coupling at
neutral pH, high salt and
over a wide temperature
range.
·
No further activation required,
easy introduction of other surface
chemistries.
Binds carbohydrates,
glycoproteins (lectins) and
glycolipids (e.g. lipopolysaccharides).
Random binding of antibodies and
C-terminal peptide-coupling.
100% covalent binding, activation
through carbodiimide is required.
Random binding of antibodies and
N-terminal coupling of peptides.
Low non-specific binding of nucleic
acids.
Coupling after 1 hour
at neutral pH and room
temperature.
Overnight coupling at
neutral to high pH and high
temperature.
5
CH3
O
Dynabeads®
M-270 Epoxy
C
H
H
H
Dynabeads®
M-270 Amine
NH2
·
·
Dynabeads®
M-270 Carboxylic Acid
COOH
2.8 µm hydrophilic beads
with surface
carboxylic acid
groups.
Covalent amide bonds ·
with primary amino
groups in proteins and ·
peptides.
·
Table 2. Product Guide - Surface-activated Dynabeads®
• Pre-coated Dynabeads®. Table 1 provides an overview of Dynabeads®
products that are pre-coupled with a linker to facilitate the binding of
specific antibodies or other common targets. The products can, for
example, be used with crude antibody solutions using a direct or indirect
purification method (please refer to chapter 2).
• Surface-activated Dynabeads ®. Table 2 provides an overview of
Dynabeads® products with different binding chemistries. These products
allow maximum flexibility for target isolation. Different surface-activated
Dynabeads® have different specific chemical functionalities allowing
direct coupling of your ligand. Which surface-activated Dynabeads® to use
depends on the nature of the ligand, and important considerations are;
the lability of the ligand, which active groups are available for coupling,
the orientation of the active site, and any hydrophilic / hydrophobic
interactions between the bead and the ligand.
www.dynalbiotech.com
61
Immediate peptide bond
formation at pH 5 and
room temperature.
Protein and Organelle Isolation
O
C
How to use Dynabeads® in Protein Isolation
Magnetic Isolation of Proteins with Dynabeads®
Magnetic separation technology is very simple with few handling steps.
Figure 3 gives an overview of the steps involved in the magnetic isolation
of proteins. Use any sample (serum, cell lysate, ascites, saliva etc) directly
without dilution. Positively isolated target proteins can be resuspended in a
small volume to maintain a concentrated protein solution. During negative
selection the sample is depleted of one or more target proteins, leaving an
untouched “negative” fraction for further studies. For technical details on
both positive and negative isolations, please refer to chapter 2.
Choosing the Size
Dynabeads® are available in three different sizes: 1.0 µm, 2.8 µm and
4.5 µm in diameter. The choice of Dynabeads® size, plus ligand, is determined
by the application. The smaller 1.0 µm (MyOneTM ) and 2.8 µm (M-270
and M-280) Dynabeads® provide greater surface area per mg beads, thus
increasing binding efficiency and capacity in most protein applications. The
larger Dynabeads® (M-450 and M-500) are ideal for isolation of cells and
organelles.
Hydrophobic or Hydrophilic?
Hydrophobic Dynabeads® enable rapid adsorption of proteins to the bead
surface with the subsequent formation of hydrophobic or covalent bonds.
Optimal orientation of some proteins, such as IgG, on the bead surface is
facilitated by the initial adsorption of hydrophobic areas of the protein.
These Dynabeads® are stable in aqueous solutions. Dynabeads® M-450,
Dynabeads ® M-280 and Dynabeads ® MyOne™ Tosylactivated are all
hydrophobic. Dynabeads® M-500 Subcellular have a special layer of polymers
and are slightly hydrophobic.
Hydrophilic Dynabeads® are designed for more gentle, covalent coupling of
molecules to the bead surface, ensuring that the functionality of enzymes
and fragile proteins is maintained. Coupling of peptides is also made easy.
The Dynabeads® M-270 product range and Dynabeads® MyOne™ Carboxylic
acid are all hydrophilic.
Fig. 3: Magnetic isolation of proteins with
Dynabeads®. The example shows immunomagnetic
protein isolation, but any ligand can be coupled
onto the beads for affinity protein capture.
62
www.dynalbiotech.com
Examples of Protein Applications
Fig. 4: Dynabeads® magnetic protein capture has been useful in a wide variety of applications.
Examples of Protein Applications
5
Using Dynabeads as a solid-phase in magnetic isolation of proteins enables
fast and easy affinity capture of pure proteins. The advantages of magnetic
isolation of proteins have been applied in a wide variety of applications
(fig. 4).
®
Immunoprecipitation and Co-Immunoprecipitation
+
Denatured
target protein
+
Y
Numerous publications document the applicability of Dynabeads® to smallscale immunoprecipitation and co-immunoprecipitation of pure proteins.
As a solid-phase matrix Dynabeads are widely used for fast and easy affinity
capture from crude samples such as cell lysates, whole blood, plasma and
ascites.
Reuse
Separation of protein complexes with Dynabeads® is also used to determine
the association between different proteins. Requiring only a single tube, the
separation process enables quantification of the isolated proteins. The speed
and simplicity associated with magnetic handling make Dynabeads® ideal for
isolation of proteins and protein complexes, with distinct advantages over
other solid-phase matrix based techniques.
“Pull-Down” of Large Protein Complexes
The large protein complexes that tend to break up with traditional column
chromatography techniques, remain intact when using the gentle Dynabeads®
separation procedure. Very rapid binding kinetics, reduced shearing forces
and the generally high protein concentration throughout the isolation ensure
that protein complexes remain intact. The study of these protein clusters is
important for understanding biochemical processes related to disease.
www.dynalbiotech.com
63
Y
Dynabeads® are available pre-coated with anti IgG antibodies, protein A,
protein G, Streptavidin or TALON ™. The proteins are captured via the beadbound ligand, facilitating rapid isolation of very pure protein. Subsequent
analysis or further characterisation can be easily performed. The elution
regime employed will depend on your specific downstream application
(see fig. 5).
Native
target
protein
Immobilised
target protein
Denaturing Elution
- SDS-PAGE for staining
and protein identification
- SDS-PAGE for western blotting
- SDS-PAGE for fluorography
Mild Elution
- Protein characterisation
- Immunisation
- Enzyme studies
- AA-sequence determination
- Crystallisation
No Elution
- Protein-protein interactions
- Enzyme studies
- Bioassays
- Immunoassays
Fig. 5 : Different elution possibilities for captured
proteins.
Protein and Organelle Isolation
The following pages show examples of application areas where Dynabeads®
have been used. For more details and references please contact Technical
Service or refer to our web-site www.dynalbiotech.com.
Examples of Protein Applications
Protein Isolation Through Tags
Dynabeads
% binding of TNF
®
Microtitre plate
time r
time (minutes)
Fig. 6: Dynabeads® binding kinetics are superior
to traditional microtitre plates. The graph
shows % binding of tumor necrosis factor to
immobilised antibody as a function of time.
Courtesy of N-B Liabakk, Department of Cancer
Research and Molecular Medicine, Norwegian
University of Science and Technology, Norway.
Pull-down of GST-fusion proteins can be performed with Dynabeads® by
applying anti-tag antibodies. Another option is to fuse the target proteins
with protein A and separate using Dynabeads® coated with an IgG that binds
to protein A .
Immunoassays
Specific antigens or antibodies immobilised to Dynabeads® ensure rapid
reaction kinetics in the binding process (fig. 6). The detection system can
be based on the use of enzymes, radioisotopes, fluorescent substances
or chemiluminescence. The exact amount of Dynabeads® used for each
IA-system must be titrated and depends on assay conditions, antibodies
used etc. (ref. chapter 7).
Phage Display Panning
Screening of phage display libraries is a valuable tool in the mapping of
protein-protein interactions, detection of diagnostic markers and identification
of novel drug candidates in drug discovery. A target protein is coupled onto
Dynabeads® and screened against a phage library. Positive binders are easily
separated from non-binding phages by magnetic separation.
Dynabeads® give an increased sensitivity compared to traditional tube or
microtitreplate panning:
• Large surface area allows for panning of up to 1013 phages/ml.
• Higher percentage of positive binders and fewer rounds of panning.
• Increased ease and thoroughness of washing.
• Magnetic separation allows for high throughput automation.
When using antibody targets, biotinylated targets and expression tags,
Dynabeads® allow for the appropriate orientation of the binding domain. The
possibility of applying different types of Dynabeads® with specific surface
functionalities for panning is also a benefit, resulting in only target specific
binders. See table 3 for examples of Dynabeads® used in phage display
panning.
Desired Binding Property
Advantage
Product
Easy coupling of target to bead through
histidine affinity-tags.
Obtains correct orientation of target for panning.
Very inert surface ensures low non-specific
binding.
Dynabeads® TALONTM
Easy coupling of biotinylated target.
Obtains correct orientation of target for panning.
Dynabeads® M-280 Streptavidin
Direct and very efficient covalent coupling
of target onto a hydrophobic surface.
Allows easy coupling of antibodies with optimal
orientation for affinity capture of proteins.
Dynabeads® M-280 Tosylactivated
Direct and very gentle covalent coupling
of target onto a hydrophilic surface.
Allows gentle binding of structurally intact and
active peptides, proteins and enzymes.
Dynabeads® M-270 Epoxy
Table 3. Examples of Dynabeads® that can be used in Phage Display Panning.
64
www.dynalbiotech.com
Dynabeads® in Automated Systems
Recombinant Protein Isolation
Dynabeads® TALONTM* (fig. 7) provides a quick and convenient method of
isolating recombinant histidine affinity-tagged** proteins through the cobalt
based TALONTM chelator on the surface of the Dynabeads®. The quality of
expressed recombinant proteins can easily be determined by elution of
isolated protein and application to mass spectrometry (fig. 8).
The pure proteins captured by Dynabeads® TALON™ can be eluted from the
beads, or used directly as solid-phase in downstream applications.
* The IMAC technology for Dynabeads® TALON™ is licensed from BD Biosciences Clontech.
** Dynal Biotech recommends the use of BD Biosciences Clontechs HAT and HN6 tags.
Fig. 7: Binding of a histidine affinity-tagged
recombinant protein (red) to Dynabeads ®
TALON™ through metal coordination of the
histidine-tag to the cobalt ion (blue) held in the
TALON™ chelator (black).
Downstream Mass Spectrometry Analysis
Dynabeads® can be used to immunoselect proteins for further separation
on SDS PAGE and downstream analysis by mass spectrometry. Biotinylated
peptides isolated by using streptavidin-coupled Dynabeads® can also be
applied directly in MALDI-TOF mass analysis.
A
5
Intellectual Property
Due to the vast number of possible applications with Dynabeads® Dynal
Biotech will not be responsible for violations or patent infringements that
may occur with the use of our products.
B
Automation
Intrinsic features of Dynabeads® make them perfectly suited for automation.
All Dynabeads® - both within and between batches - are identical in size,
shape, surface properties and iron content. Although the beads disperse well
and sediment slowly (fig. 9), in a magnetic field they move quickly and with
an even pull towards the magnet. This facilitates rapid target binding, as well
as short incubation and separation times. No mixing or stirring is necessary.
The Dynabeads® do not aggregate, ensuring homogeneous fluid behaviour
in automated systems, without any blocking or clogging of pipette tips.
Dynabeads® act as a pipettable solid-phase which handles like a liquid.
Fig. 8: Mass spectrometry spectra and reconstructed (deconvoluted) mass diagram on purified
and desalted recombinant protein.
A: Raw TOF MS spectrum (single scan) of the
protein after bead clean-up.
B: Reconstructed spectrum obtained from A by
MaxEnt1 deconvolution.
Courtesy of Johanna Steen et al., KTH, Royal
Institute of Technology, Stockholm, Sweden.
A range of Dynabeads® with one micron diameter are made especially for
automated protocols. Dynabeads® TALON™ for binding of histidine affinitytagged proteins, Dynabeads® MyOne™ Streptavidin C1 for binding of
biotinylated proteins and Dynabeads® MyOne™ Tosylactivated for binding
of any ligand recognising your protein.
Automated protocols are available for Dynabeads® TALON™ on KingFisher®
(Thermo Electron), Tecan Genesis and Freedom EVOTM (Tecan AG), Biomek FX®
(Beckman Coulter) and Magnatrix 1200TM (Magnetic Biosolutions).
Fig. 9: Analysis of the sedimentation rate for
Dynabeads® TALONTM.
For more information see www.dynalbiotech.com.
www.dynalbiotech.com
65
Protein and Organelle Isolation
Deconvolution
Isolation of Pure Organelles
Subcellular Fractionation with Dynabeads®
Subcellular fractionation is traditionally performed by density gradient
centrifugation of cell homogenates. This technique is time-consuming and
labour-intensive, and has several clear limitations. As different organelles
do not have distinct size and density definitions, fraction losses and
mixed fractions are common problems with techniques based on these
attributes.
Fig. 10: Due to their ultra-smooth surface, the
Dynabeads® M-500 Subcellular are well suited for
the isolation of organelles, vesicles or membranes
for EM-studies. Courtesy of Dr. KE Howell,
Department of Cellular and Structural Biology,
University of Colorado, Denver, CO, USA.
In contrast, the Dynabeads® immunomagnetic isolation technique separates
the organelles into fractions based upon their immunological properties,
avoiding the problems associated with separating organelles of similar sizes
and densities. Electron microscopy of isolated vesicles or membranes for
morphometric studies can conveniently be performed using the ultra-smooth
Dynabeads® M-500 Subcellular (figs. 10 and 11).
As more and more organelle specific markers are identified, corresponding
immunomagnetic isolation of subgroups of organelles can be performed.
Examples of organelles or organelle fractions that are isolated with
Dynabeads® include; microsomes, tubulovesicles, plasma membrane
vesicles, mitochondria, osteoclast membranes, peroxisomes, (fig. 12)
endosomes, phagosomes, golgi, synaptic vesicles, TGN vesicles, exosomes,
caveolae, liposome fused microsomes, GLUT4 vesicles and lysosomes.
When analysing the total organelle specific protein population for proteome
investigations, performing subcellular fractionation as a preparative step
gives a range of advantages, including:
Fig. 11 : Dynabeads® M-500 Subcellular coated
with polyclonal pig anti-rabbit antibodies and
then with rabbit antibodies specific for various
proteins. The figure shows vesicles bound to antilactase coated beads (A and D), to anti-galectin-4
coated beads (B and E) and control beads with
only the linker antibody, no vesicles bound (C and
F). First published in J. Biol. Chem.
Courtesy of Gert H. Hansen and E. Michael
Danielsen, The Panum Institute, University of
Copenhagen, Denmark.
•
•
•
•
•
Reduced complexity
Assessment of low abundance proteins
Isolation of dynamic proteins
Compatibility with downstream applications
Sorting with direct link to protein functionality
The Product of Choice for Subcellular Fractionation
Dynabeads® M-500 Subcellular, different pre-coated Dynabeads® M-450 and
some of the Dynabeads® M-280 products, are widely used for the isolation
of subcellular fractions. Dynabeads® M-500 Subcellular (supplied as 4 x 108
beads/ml) are optimised for immunomagnetic fractionation of subcellular
compartments, and due to the ultra-smooth surface of this product they
are perfect for imaging via electron microscopy. Dynabeads® M-500 are
also less prone to non-specific binding due to their weak hydrophobicity.
Primary antibodies can also be coupled to anti-Ig coated Dynabeads ®
(see chapter 3). The size and iron content of these beads ensure complete
isolation of organelles or whole cells, even from viscous starting samples.
For smaller targets, such as proteins, the smaller Dynabeads® M-280 are
recommended. For isolation of organelles using ligands other than antibodies
the Dynabeads® M-270 Epoxy and Dynabeads® M-280 Tosylactivated can be
used for high purity organelles. Further specifications are listed in table 4.
For a full reference list please see www.dynalbiotech.com or contact our
Technical Centre.
66
www.dynalbiotech.com
Isolation of Pure Organelles
Overcoming the Limitations of Other Techniques
Used alone, density gradients have some clear limitations:
• When separating a heterogeneously sized organelle population by density,
some losses will occur. This is especially important in the study of some
diseases, where the size and density (and therefore also sedimentation
coefficient) of the affected organelles will vary as a result of changed
physical conditions (Lüers GH et al. Immunoisolation of highly purified
peroxisomes using magnetic beads and continuous immunomagnetic
sorting. Electrophoresis 1998;19:1205-1210).
• When separating by density, organelle fractions possessing similar
densities (e.g. microsomes and peroxisomes) will be isolated in the same
fraction, resulting in the membranes being obtained as a mixture rather
than as a pure population (Sexton PS and Cenedella RJ. Immunomagnetic
capture of lens membrane fractions containing steroid binding protein.
Biochem. Biophys. Res. Commun. 2002;295(4):1027-1031).
• Limitations of detergent extractions resulting in mixed fractions can be
avoided using Dynabeads® (Sans N et al. Synapse-Associated Protein
97 Selectively Associates with a Subset of AMPA Receptors Early in their
Biosynthetic Pathway. J. Neurosc. 2001;21(19):7506-7516).
5
Application Guide
Application
Antibody
Recommended product
Comments
EM pictures.
Any antibody
Dynabeads® M-500 Subcellular
Super smooth surface enables
superior EM pictures of isolated
organelles.
Isolation of fragile
organelles or organelle
fractions in general.
A mouse monoclonal
antibody.
Dynabeads® M-280 Sheep anti-Mouse IgG
Direct or indirect isolation of
organelles made more efficient
with two layers of Abs.
A rabbit antibody.
Dynabeads® M-280 Sheep anti-Rabbit IgG
Direct or indirect isolation of
organelles made more efficient
with two layers of Abs.
Antibodies binding to
protein A or protein G.
Dynabeads® Protein A
Dynabeads® Protein G
Efficient direct or indirect isolation
of organelles.
Dynabeads® M-450 with a secondary
antibody against the corresponding primary
Ab species.
The larger M-450 Dynabeads®
ensure a better pull to the magnet
in viscous samples.
Isolation of large
Antibodies made in a
organelle fractions or
specific species.
isolation from a viscous
sample.
Table 4: Application Guide – Subcellular Fractionation
www.dynalbiotech.com
67
Protein and Organelle Isolation
F ig. 12: Perox isomes isolated with, and
photographed while attached to, Dynabeads®
Cour tesy of Prof. A . Völkl, Universit y of
Heidelberg, Germany.
Ordering Information
Ordering Information
Product
Volume
Product No.
Dynabeads® TALONTM
40 mg/ml
2 ml
10 ml
101.01
101.02
Dynabeads® Protein A
~ 40 mg/ml
1 ml
5 ml
100.01
100.02
Dynabeads® Protein G
~ 30 mg/ml
1 ml
5 ml
100.03
100.04
Dynabeads® M-280 Sheep anti-Mouse IgG
10 mg/ml
2 ml
10 ml
112.01
112.02
Dynabeads® M-280 Sheep anti-Rabbit IgG
10 mg/ml
2 ml
10 ml
112.03
112.04
Dynabeads® M-280 Streptavidin
10 mg/ml
2 ml
10 ml
100 ml
112.05
112.06
602.10
Dynabeads® MyOne™ Streptavidin C1
10 mg/ml
2 ml
10 ml
100 ml
650.01
650.02
650.03
2 ml
10 ml
100 m
656.01
656.02
656.03
Dynabeads® M-450 Epoxy
30 mg/ml
10 ml
100 ml
302.02
302.03
Dynabeads® M-270 Epoxy
60 mg
300 mg
freeze-dried
freeze-dried
143.01
143.02
Dynabeads® M-450 Tosylactivated
30 mg/ml
10 ml
302.12
Dynabeads® M-280 Tosylactivated
30 mg/ml
30 mg/ml
100 mg/ml
2 ml
10 ml
10 ml
142.03
142.04
301.01
Dynabeads® MyOne™ Tosylactivated
100 mg/ml
2 ml
10 ml
655.01
655.02
Dynabeads® M-270 Carboxylic Acid
30 mg/ml
2 ml
10 ml
143.05
143.06
2 ml
10 ml
100 ml
650.11
650.12
650.13
2 ml
10 ml
143.07
143.08
2 ml
150.01
Dynabeads® MyOne™ Streptavidin T1
10 mg/ml
NEW
Dynabeads® MyOne™ Carboxylic Acid
10 mg/ml
Dynabeads® M-270 Amine
30 mg/ml
Dynabeads® M-500 Subcellular
30 mg/ml
68
www.dynalbiotech.com