Biology Reference
In-Depth Information
9. If the pH is above 8.2, use lysis buffer adjusted to pH 6.5
accordingly. pH readjustment using lysis buffer is preferable to
adjustment by NaOH or HCl, since the concentration of the
chaotropic salts is maintained at the optimal level.
1. Carefully determine the total protein concentration of the
lysate using a detergent compatible assay (saturation DIGE
manufacturer GE Healthcare recommends Protein Deter-
mination Reagent USB, code 30098). Do not change the assay
throughout the saturation DIGE experiment since assays of
different type and manufacturer may lead to different results.
2. If the concentration matches the recommended range
(>0.55
3.2. Determination
of Total Protein
Concentration
L), but a dilution is required for techni-
cal reasons, this may be performed by addition of lysis buffer.
3. In contrast, if the concentration is not within the recommended
range, prepare a new lysate using an adequate sample amount
and/or lysis buffer volume.
μ
g/
μ
L, <5
μ
g/
μ
3.3. Optimization
Experiment:
Development
of a Sample-Specifi c
Labeling Protocol
The maleimide derivatives of saturation CyDyes react with free
sulfhydryl groups, and consequently, proteins to be included in
the analysis must contain at least one cysteine residue. Additional
cysteines lead to a proportional increase in fl uorescence signal
intensity of the affected protein. If present in excess amount,
CyDyes also react with primary amine groups, leading to one or
even more additional labels of the protein at lysine residues. Since
the positive charge of each affected lysine residue is eliminated, the
pI of the molecule becomes more acidic. As a consequence, two
or even more derivatives of the protein are generated, leading to
additional 2D gel spots along the horizontal (pI) axis of the affected
protein. This effect is referred to as “overlabeling.” In contrast, an
insuffi cient concentration of CyDye will fail to label all cysteine
residues in the lysate to completion. Since sulfhydryl groups are not
charged at neutral or mild acidic or basic pH, no horizontal shift
will occur. Instead, especially in cysteine-rich small proteins, the
lower molecular mass of unlabeled cysteine residues may be resolved
by 2D gel electrophoresis, leading to additional spots in the vertical
(molecular mass) axis. The stoichiometric conditions facilitating
complete labeling of all cysteine residues without affecting lysine
residues have to be determined individually in a titration experiment,
since (1) content of sulfhydryl-containing biological compounds
(e.g., glutathione) may vary considerably between organisms,
organs, cells, and body fl uids (see Note 7) and (2) individual sample
preparation conditions may lead to the introduction of exogenous
interfering chemical compounds (traces of reducing agents, free
primary amines, etc.). Whenever the biological source, the cell
culture conditions, or the sample preparation protocol is changed,
the optimization step must be repeated. Within one experimental
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