Bio-Nobile's PickPen® magnetic
tool and QuickPick™ kits
Each of Bio-Nobile’s QuickPick™ protein kits contain reagents and validated
protocols for a particular method, or chromatography chemistry. The kits are
optimized for use with the PickPen® magnetic tool, and are
especially designed for small sample amounts with fast and sample-conserving
analysis in mind. While conventional methods can often take several hours, QuickPick protein kits reduce protocol time to 5 minutes.
Different size versions of the kits are available, and they include sufficient reagent for
either 8 or 48 preparations.
Methods are currently available for
Major benefits of PickPen methods
Fast
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You move particles and not liquids, and avoid time-consuming
aspirating and dispensing stages |
Easy
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PickPen is easy to learn, easy-to-use and easy-to-maintain
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Efficient
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You no longer need to share equipment, you can work with
your own choice of vessel formats, and you have full visual control of the
purification process |
Gentle
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You can maintain high sample quality through rapid handling
without multiple centrifugations and spin columns |
Ion exchange methods (CM and DEAE)
Proteins consist of many different amino acids,
and the overall charge is caused by the composite effect of many different
ionizable groups. The pH at which the protein has no net charge is called the
isoelectric point, and is termed pI. The pI of most proteins is in the range of
5-9. Ion-exchange of proteins is typically performed at least 1 pH unit away
from the pI of the protein of interest. When the pH is below the pI, the
molecule will be positively charged and a cation exchange resin should be used.
When the pH is above the pI, the molecule will be negatively charged and an
anion exchange resin should be used. Since interactions of ion-exchange groups
with proteins depend on the surface charges of the protein, even a protein at
its pI may bind to the ion exchange matrix.
In the purification of proteins, a low buffer concentration must be used during
binding. Otherwise the buffer components will compete with the protein for the
exchange sites.
Low buffer strength
If the concentration of the buffer
ions is high, then the buffer ion will tend to bind to the ionic sites on the
particles and any bound target will tend to elute:
High buffer strength
The weak ion exchange methods in
Bio-Nobile QuickPick kits are based on magnetizable
cellulose particles coated with carboxymethyl (CM) or diethyl aminoethyl (DEAE)
groups. Different types of proteins will bind to the CM or DEAE coated magnetic
particles with affinities that depend on both the conditions used and the types
and number of individual charged groups. In typical protein purification using
ion exchange chromatography a mixture of many proteins is used as a sample.
After unbound molecules are washed away conditions can be stepwise adjusted,
such as by altering the pH and salt concentration to release the molecule(s) of
interest from the particles.
QuickPick CM or DEAE protocol - all magnetic particle transfers are carried out with
PickPen
Pipet the reagents into tubes 1-7 as follows:
- tube 1: Magnetic Particles
- tube 2: Regeneration Buffer
- tube 3: Wash Buffer
- tube 4: sample
- tubes 5 and 6: Wash Buffer
- tube 7: Elution Buffer
Regenerate and wash Magnetic Particles, then transfer to sample tube and
incubate for 2 minutes at room temperature. Proteins anneal to the Magnetic
Particles.Wash Magnetic Particles twice with Wash Buffer.
Elute proteins from the Magnetic Particles for 1 minute at room temperature.
Collect the Magnetic Particles from the elution tube and discard them.
Supernatant now contains the proteins and is ready for further analysis. |
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High quality purifications using ion exchange methods
Not only do PickPen methods allow high quality purifications, they also allow
flexibility in working methods. You can use the PickPen magnetic tool to
concentrate a protein. This is illustrated below for
the protein, cytochrome c.
Cytochrome c in I mg/ml solution captured on magnetic particles using the
QuickPick CM kit and resuspended in a smaller
volume.
| Sample containing cytochrome c |
Cytochrome c removed |
Cytochrome c resuspended |
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| 150 μl |
150 μl |
25 μl |
Immobilized metal ion affinity chromatography (IMAC)
Immobilized metal ion affinity
chromatography (IMAC) was introduced by Porath et al. in 1975 as a method for
group separation, but today it represents one of the most important tools for
the single-step purification of proteins based on polyhistidine tags. The
principle of the mechanism is based on the interaction between a metal ion
coordinated to a covalently bound chelating ligand with histidine-containing
proteins. The selectivity of the adsorption in IMAC increases, whereas the
amount of bound protein decreases, when immobilized metal ions are used in the
following order: Cu2+, Ni2+, Zn2+ and Co2+. The use of the weakest metal ion
that still binds the protein of interest is the preferable choice because it
will result in the smallest amount of adsorbed unwanted proteins.
QuickPick IMAC magnetic particles are formed from magnetizable agarose particles
which affords an extensive porous surface area especially preferred in the
purification of histidine tagged proteins from cell lysates. The transfer of
magnetic particles with the PickPen magnetic tool enables rapid purification of histidine tagged proteins with suitable binding and elution characteristics. The
methodology used abolishes the need for sample pre-treatment or use of
time-consuming column technology.
QuickPick IMAC protocol - all magnetic particle transfers are carried out with PickPen
Pipet reagents into tubes 1-6 as follows:
- tube 1: Magnetic Particles
- tube 2: Regeneration Buffer
- tube 3: Wash Buffer 1
- tube 4: sample
- tube 5: Wash Buffer 2
- tube 6: Elution Buffer
Regenerate and wash with Wash Buffer 1 the Magnetic Particles and then
transfer to sample tube and incubate for 2 minutes at room temperature. The
His-tagged protein anneals to the Magnetic Particles.
Wash the Magnetic Particles with Wash Buffer 2.
Elute the His-tagged protein from the Magnetic Particles for 1 minute at
room temperature.
Collect the Magnetic Particles from the elution tube and discard them.
Supernatant now contains the His-tagged protein and is ready for further
analysis.
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High quality purificatins using QuickPick IMAC
The performance of the QuickPick IMAC kit in purifying His-tagged
protein specifically is illustrated by the example below in which
E.coli His-tagged glutathione-S-transferase (HT-GST) was isolated
using the PickPen/QuickPick method.
The cells were lysed in 20 mM Tris-HCl buffer, pH 7.0 before
purification.
Purity checked by analyzing 5, 7.5 and 10 µl of eluate on SDS-PAGE gel. The HT-GST protein band is clearly seen for each
of the three eluate lines.
M = Molecular weight markers
1 = cell lysate
2 = lysate after MP treatment
3 = 5 µl QuickPick IMAC eluate
4 = 7.5 µl QuickPick IMAC eluate
5 = 10 µl QuickPick IMAC eluate
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