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Frequently asked questions about protein purifications
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What applications do you have for proteins?
- I would like to purify specific proteins from plant or other
source. How could I apply your technology?
- My his-tagged protein is expressed as insoluble inclusion bodies. Can I
purify them with your technology?
- Is it possible to regenerate the magnetic particles used in protein
purification kits?
- After purification some of the target protein remained in the sample. Can I
use the sample once again?
- Can you give me information about the binding capacity of the protein kits?
- Does IMAC work with high salt concentrations?
For protein purifications we have different approaches for the purification
strategy. First, we have fractionation kits such as QuickPick™ DEAE (using weak
anion exchange) and QuickPick CM (using weak cation exchange). By using these
kits you are able to enrich your target protein to a fraction containing also
other proteins. This may be a good choice when you have a complex protein sample
and need to do a prepurification step before actual purification. Secondly there
are kits based on affinity chromatography such as IMAC (immobilized metal
affinity chromatography) and GST (glutathione-S-transferase). IMAC affinity
purification is based on the specific separation of histidine-tagged proteins and
is one of the most widely used techniques up-to-date. Using Bio-Nobile’s
QuickPick IMAC Plus kit it is possible to purify up to 0.5 mg protein per
preparation. The QuickPick GST glutathione affinity kit is intended for the
purification of GST-fusion proteins. At least 40 µg of GST-fusion protein can be
purified per preparation. All the protein kits are ready-to-use containing all
the reagents needed for the purifications.
There are several approaches:
a) If you want to purify specific proteins from plant or other source you would
need to have the specific Abs against them. In this case and if you will label
the Abs with biotin you could use our Streptavidin magnetic particles for
protein purification.
b) We also have magnetic particles based on ion-exchange properties. These would
give you an opportunity to fractionate the proteins and enrich your target
proteins to a certain level. You would need to have an analysis method (e.g.
enzymatic, Western blot etc) for the identification of the correct target
protein.
c) The next possibility is to clone the genes of the proteins (if you know their
DNA sequences) in a His-tag or GST protein encoding expression vector and make a
His-tag or GST fusion proteins out of them. In this case you could use our IMAC
or GST kits for the purification of the proteins.
Yes, by using our QuickPick IMAC or IMAC Plus kit the purification can be
performed under strongly denaturing conditions in which the inclusion bodies are solubilized. A technical note for the protocol
may be downloaded from our literature
pages.
The magnetic particle technology for protein purification is based on typical
group specific and affinity chromatographic chemistries. The magnetic particle
can be regenerated using conventional regeneration methods used in
chromatography. However, Bio-Nobile does not guarantee that results obtained by
using regenerated magnetic particles are within the specifications defined for
the kits.
One of the great benefits of Bio-Nobile’s technology is that the sample remains
for additional use, if needed. In this case you may repeat the purification with
fresh reagents to obtain the rest of the target protein. If a concentrated
target protein solution is needed the protein can be eluted in the same elution
buffer that was used for the first purification.
The binding capacities per preparation in our protein kits are as follows:
QuickPick CM: 60 µg ± 6 µg (aprotinin)
QuickPick DEAE: 60 µg ± 6 µg (BSA)
QuickPick IMAC: 30 µg ± 5 µg (His6-glutathione-S-transferase)
QuickPick IMAC Plus: 0.5 µg ± 50 µg (His6-glutathione-S-transferase)
QuickPick GST: 40 µg ± 4 µg (GST)
In IMAC metal affinity method the salt concentration plays no role in binding
His-tagged proteins. High salt concentration in sample might prevent unwanted
proteins from binding to the particles. Instead the concentration on Imidazole
is important: in low imidazole concentration (~5 mM) the His-tagged proteins
bind to the particles. Then the particles are washed in a bit higher imidazole
concentration (5-40 mM) to get rid of all the other proteins. And in high
imidazole concentration (>300 mM) the His-tagged proteins will detach from the
particles.
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