Biology Reference
In-Depth Information
buffer accelerates solubilization kinetics, though overall
solubility of a protein remains unaffected. Choose your lysis
buffer volumes appropriately so the minimum protein concen-
tration is >0.55 mg/mL.
6. pH of volumes as low as 0.5
L can be measured using a narrow
range pH indicator strip. However, since the color references
tables of the manufacturer refer to indicator strips completely
saturated with the solvent to be measured, a signifi cant color
deviation may occur when only submicroliter volumes are
applied. To avoid erroneous pH measurement, apply on the
same indicator strip in the appropriate fi eld an equal amount of
fresh lysis buffer, adjusted to pH 8.0 with a calibrated pH
meter. The resulting color of the original buffer is your unbi-
ased reference. Should readjustment of your sample pH be
necessary, proceed in the same manner, preparing a fresh refer-
ence spot each time.
7. Assuming a protein concentration of 5
μ
L in the protein
lysate and 1% of all amino acids represent cysteine, the concen-
tration of cysteine-derived SH groups in the lysate equals
420
μ
g/
μ
mol/L. Calculating with a glutathione concentration of
5 mmol/L in mammalian tissue and 25
μ
g protein extracted/
mg tissue, the concentration of glutathione-derived SH groups
in the lysate is 1,000
μ
mol/L, e.g., in the same range as
cysteine-derived sulfhydryl groups. Therefore, varying amounts
of GSH or other sulfhydryl-containing compounds in individ-
ual samples may considerably alter the overall sulfhydryl
content in the lysate.
8. Labeling procedures for analytical gels are described for a total
protein amount of 5
μ
g, as recommended by the manufacturer
(GE Healthcare). According to our experience, the amount
can be further decreased if necessary. In any case, develop the
sample labeling protocol with the same amount of protein as
will be used in the fi nal experiments.
9. According to our experience, not all the material is necessary
to produce excellent gel images. Consider to use only half the
amount (e.g., 2.5
μ
g Cy5 labeled material per
gel) in order to be prepared for a potential repetition of the 2D
PAGE analysis.
10. If all experimental procedures are carried out carefully, the
variations introduced by the biological system will usually
exceed the variations introduced by the experimental setup.
Thus, it is much more effective to perform biological replicates
than technical replicates, since the biological replicates neces-
sarily comprise all the technical variations, whereas technical
replicates do not comprise any biological variations.
11. As an example, analyzing effector-induced proteome changes
at four time points (t1, t2, t3, t4) in two cell lines (A, B), six
μ
g Cy3 and 2.5
μ
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