Biology Reference
In-Depth Information
3. Determine mass spectra and MS/MS fragment ion mass with a
4800 MALDI TOF/TOF analyzer.
4. Submit all product ions to a computer database search analysis
with the Mascot MS/MS ion search.
4
Notes
1. Appropriate extraction buffers for plant specimens or allergens
should be selected. In general, soluble buffers such as PBS,
Tris-HCl or NaCl solution are used as the extraction buffer.
Commercial protein extraction kits for plant specimens may
also yield good results within a short time. Prior to the
2D-immunoblotting, it should be confi rmed by ELISA or
immunoblotting using the patients' sera, that the extracts con-
tain allergens.
2. After addition, vortex well and allow standing until the resins
are completely settled. Do not use if the powder color changes
to yellow, or the saturated solution is not clear.
3. Careful attention should be paid to desalt samples, since a
high-salt buffer may be used for extracting the allergens. Most
failures in spot focusing may be caused by not paying suffi cient
attention to this step.
4. Move the strip up and down so that entire surface of the gel
comes in contact with the sample solutions.
5. Slowly drop the oil from the tip of the holder. Excessively rapid
dropping of the oil would cause the samples to fl oat above the
gels. Be careful to avoid fl ooding with oil from the side of the
holder.
6. We set long-time runs at low voltage so that any salt contami-
nating the samples moves away to the tip of the holder. It
would not be a problem even if the voltage did not reach up to
8,000 V at the end of the run. If the voltage does not rise at all
during the fi rst step, it could be assumed that the sample prep-
aration or rehydration has not been satisfactory.
7. Addition of buffers to a frozen strip might cause incomplete
reduction and/or alkylation. Put the side of the strip on the
bottom of the dish so that the gels get completely fl ooded by
the buffer.
8. Cut 5 mm off both sides of the plastic sheet on the gels as to
obtain a good fi t into the gel slot. Place the plastic side of the
strip on the longer plate and put it on the acrylamide gel with
the fl at side of a spatula. Be careful not to put it into the gels.
If air bubbles are present between the strip and gel, remove
them with a spatula.
