Biology Reference
In-Depth Information
4. Notes
1.
To protect samples against tissue disruption (freezer burn)
after resection the tissue is placed in tin foil at fi rst and then
stored in a cryo-tube.
2.
Eosin has an adverse effect on protein recovery and should be
omitted (
3 ). An alternative stain compatible with the proce-
dure is methylgreen ( 10 ) .
3.
First, count for each cell type to be harvested the required
number of cells (e.g., 5,000) in series of slices and measure the
area the cells occupy. These areas can be used to estimate the
required cell numbers during routine microdissection.
4.
Usually, depending on the IEF tube's diameter, not more than
50
L can be applied. Therefore, we increased the volume
by blowing up the glass tube so that a sample volume of
100-200
μ
μ
L can be applied.
5.
Prior to labeling, the pH of the sample should be checked by
spotting small drop of lysate on a pH indicator strip. A pH
below 7.8 or above 8.2 should be adjusted to 8 by adding lysis
buffer (7 M urea, 2 M thiourea, 4% (w/v) CHAPS, and 30 mM
Tris-HCl) preadjusted to pH 9.5 or 7.5, respectively.
6.
Before scanning, the glass plates have to be cleaned thoroughly.
First of all, use ethanol for removing of acrylamide or silicone
and then clean the glass plates with water.
7.
To avoid the detection of dust particles and artifacts as protein
spots in the gel, an exclusion fi lter should be applied. We
recommend excluding all spots with a volume <30,000.
Alternative software solutions, e.g., Delta 2D (Decodon) or
Progenesis SameSpots (Nonlinear Dynamics), can also be used
for image analysis. Each software package has its pros and cons,
e.g., no missing value, spot co-detection features, or alterna-
tive options for statistical analysis ( 11 ) .
8.
Air bubbles should be avoided by applying the solution slowly
under the surface (1 mm) of the solution which has been
applied before.
9.
To prevent destruction of the IEF gel by the nylon fi ber, (a)
the thermoplastic nylon fi ber should be fi tted to the tube inner
diameter by melting one end into the tube and (b) the gel
should be extruded using the cap gel (acrylamide concentra-
tion of 12.3%) as a cushion or (c) another possibility is to
polymerize a highly concentrated acrylamide solution (15%)
above the extruding side after the IEF has been fi nished and
use this gel piece as a cushion.
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