Biomedical Engineering Reference
In-Depth Information
protein separation, a gel with a proper concentration that is dependent on
the molecular weight of a target protein should be used for electrophoresis.
Generally, low-concentration gels are suitable for high molecular weight pro-
teins whereas high-concentration gels are suitable for low molecular weight
proteins. Alternatively, a gel with gradient concentrations can be useful for
properly separating proteins with a wide range of molecular weights.
Protein transfer
The purpose of transferring proteins from a polyacrylamide gel to an absor-
bent membrane is to securely localize the separated proteins for immuno-
identifi cation. To produce an identical replica of a gel on a membrane
for western blotting, effi cient transfer of proteins is required. The protein
transfer effi ciency depends on properties of gels and membranes, such as
the pore size and thickness of a gel, and the material type and pore size
of a membrane. Generally, the effi ciency increases with the pore size but
decreases with the thickness of a gel. Nitrocellulose and polyvinylidene
difl uoride (PVDF) are effective absorbent membranes commonly used for
protein transfer (Bio-Rad, 2010). Nitrocellulose has a high protein bind-
ing capacity (80-100 µg/cm
2
) but is diffi cult to handle due to its brittleness.
PVDF membranes, on the other hand, are physically strong and also feature
a higher protein binding capacity (170-200 µg/cm
2
) and a long shelf life.
The major concern of using a PVDF membrane for immunoblotting is that
PVDF is extremely hydrophobic and needs to be completely wet before
use. The composition of transfer buffer can affect transfer effi ciency as well.
The addition of methanol in a buffer solution increases binding of proteins
onto a membrane but decreases mobility of proteins moving from a gel to
a membrane during protein transfer. In contrast, the addition of SDS deter-
gent decreases binding but increases mobility of proteins. Finally, transfer
effi ciency can be affected by the method by which the proteins are trans-
ferred; protein transfer using semi-dry blotting is faster than wet blotting.
However, wet transfer produces better uniformity of transfer for certain
applications (Promega, 1997). Overall, to obtain the best result of gel blot-
ting, optimal variables, such as membrane properties, transfer time, applied
voltage and use of detergent and methanol, should be empirically optimized
and then applied for protein transfer. After proteins are transferred to a
membrane, success of the transfer process can be verifi ed by staining the gel
with coomassie brilliant blue or simply examining if the protein standard is
transferred onto the membrane.
Protein identifi cation using immuno-detection
To detect a target protein, a blotted membrane is fi rst labeled with the
target protein-specifi c antibody (primary antibody), and then with the
