Biology Reference
In-Depth Information
4
Notes
1. Cell lysis can be achieved by different methods. Plant cells pos-
sess tough cell walls that are diffi cult to disrupt. The mortar
and pestle procedure described above is usually the method of
choice for plant tissues but ultrasonic disintegrators and
homogenizers (e.g., blender) can be also used. Proceed as
quickly as possible and avoid the homogenate to thaw.
2. Tris buffer can be added when basic conditions are required
for full solubilization or to minimize proteolysis.
3. Detergents help to disrupt membranes, solubilize lipids, and
delipidate proteins bound to vesicles or membranes. CHAPS
is especially useful in purifying
membrane proteins
and can be
used in combination with other
nonionic
detergents such as
Triton X-100
.
4. PVPP-bound polyphenols are removed during the centrifuga-
tion step.
5. During cell lysis, proteases are released or activated. Protease
inhibitors minimize protein degradation during protein extrac-
tion and solubilization. The plant protease inhibitor cocktail
used contains a mixture of molecules with broad specifi city for
the inhibition of serine-, cysteine-, aspartic-, and metallopro-
teases, and aminopeptidases. Protease inhibitors will be dis-
played in 1-D and 2-D polyacrylamide gels.
6. These columns contain Sephadex™ G-25 medium. They can
be used in a wide range of applications such as desalting, buf-
fer exchange, and cleaning up of samples.
7. The mixture TCA/acetone is more effective in precipitating
proteins than either TCA or acetone alone.
8. Desalting can be achieved by gravity or centrifugation. There
is a slightly higher recovery and desalting capacity using grav-
ity protocol (described above) but sample is diluted 1.4 times.
9. Overnight precipitation results in a higher protein recovery.
10. This step is critical. An insuffi cient drying will result in an
incomplete acetone removal, whereas protein pellets will be
hardly resuspended and solubilized after an extended drying.
11. In this step, proteins can be concentrated using low volumes
of solubilization buffer.
12. Insoluble particulates cause smearing and block gel pores
during 2-D electrophoresis.
