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FAQ
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FAQ
Does the sample have to be in a specific buffer
in order to be treated with the ProteoHook™
2DE kit?
Samples may be prepared by the method of choice prior to
treatment with the ProteoHook 2DE kit. Tris and other
amine-containing reagents should not be used. EDTA is not
recommended. Commonly used solubilization agents such as urea,
thiourea and detergents including CHAPS, SDS and DTT, may be used.
Harsh cell lysis conditions, such as boiling in SDS, may also be
used. Proteins must, however, be suspended in at least 100 mM Hepes
at pH 8.0. Samples must be at pH 8.0. (The pH of the sample should
be checked with pH paper after addition of 100 mM Hepes pH 8.0.
Further addition of 1 M Hepes, pH 8.0 may be necessary for some
samples in order to adjust the pH to 8.0.)
Can you give examples of protocols for cell lysis that have been
used with the ProteoHook kit?
Whole cell extract from Drosophila embryos: Transfer
embryos to a cold ground glass homogenizer on ice. Add lysis buffer
containing 8 M urea (or 7 M urea and 2 M thiourea), 2% CHAPS, 10 mM
DTT and 100 mM Hepes pH 8.0. Homogenize. Transfer to a
microcentrifuge tube and spin briefly. Transfer the supernatant to a
clean tube.
Whole cell extract from bacterial cells: Harvest cells by
centrifugation at 5000xg. Wash cell pellet with cold 100 mM Hepes,
pH 8.0. Transfer cell pellet to a cold ground glass homogenizer on
ice. Homogenize with lysis buffer containing 8 M urea (or 7 M urea
and 2 M thiourea), 2% CHAPS, 10 mM DTT and 100 mM Hepes pH 8.0.
Transfer to a microcentrifuge tube and spin briefly at high speed.
Transfer the supernatant to a clean tube.
Whole cell extract from mammalian cells: Wash cells with cold
PBS. Transfer cells to a centrifuge tube. Add lysis buffer
containing 8 M urea (or 7 M urea and 2 M thiourea), 2% CHAPS, 10 mM
DTT and 100 mM Hepes pH 8.0. Agitate well. Spin at high speed and
transfer supernatant to a clean tube.
What sample sizes can be used with the ProteoHook 2DE kit?
Protein samples must contain 50-500 µg protein in 100-200 µl
total volume, with the concentration of protein being at least 0.5
mg/ml.
Is treatment with the ProteoHook 2DE kit compatible with 2D-DIGE?
 Yes.
Labeling samples for 2D-DIGE can be carried out at the same time as
reaction with ProteoHook reagent. Excess dye can be quenched before
capture on the ProteoHook resin. We do not recommend labeling after
treatment with the kit.
The figure to the right shows a 2D-DIGE
experiment where Drosophila embryo extracts spiked with varying
amounts of BSA were treated with the ProteoHook kit using the
suggested labeling protocol, and compared; one sample contained half
the amount of BSA compared to the other. An overlay of the Cy3 and
Cy5 images from the 2DE gel is shown. Unchanging protein spots
appear yellow. The BSA spots (marked by the white box) appear
reddish because they were present in twice the amount in the
Cy5-labeled sample.
What buffer is the treated sample in?
The treated sample is in Proteopure's proprietary elution buffer
which is compatible with 2DE. Samples can be loaded on a 2DE gel
after addition of ampholytes. We recommend using 1% ampholytes and
carrying out isoelectric focusing for no more than 40-50K volt
hours. IEF strips of the desired pH range can be used. The strips
may be rehydrated in standard buffers such as 8 M urea, 2% CHAPS, 10
mM DTT and 1% ampholytes.
Can the ProteoHook 2DE kit be used for cleanup of samples prior
to mass spectrometry?
The ProteoHook 2DE kit has been optimized for cleanup of samples
prior to 2DE. The kit includes buffers that are best suited for this
application. If you wish to try the kit for mass spectrometry, we
may be able to provide you with different buffers that are devoid of
chaotropes and detergents. Please call or email us.
Can the ProteoHook 2DE kit be used for native proteins?
The ProteoHook 2DE kit includes buffers that contain chaotropes
and detergents that are compatible with 2DE. If you wish to try the
kit on native proteins, we may be able to provide you with different
buffers that are devoid of chaotropes and detergents. Please call or
email us. Whether functionality of the protein is maintained will
depend on your specific protein.
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