2-2
Semidry Electroblotter
Thermo Scientific
Section 2
Setting Up
Materials Needed
(continued)
Blotting buffer
- The most commonly used buffer for protein blotting
from polyacrylamide gels is Towbin buffer. Small amounts of buffer may be
needed for equilibrating the gel and membrane prior to blotting, in
addition to the buffer in the transfer tank. Should be cooled to 40°C.
Filter paper
- Sometimes called blotting paper, it used in the blotting
sandwich.
Blotting membrane
- Nitrocellulose and PVDF (Polyvinylidene difloride)
can be used for proteins, while charged Nylon membranes can be used for
nucleic acids. The choice depends upon the user's preference and
sometimes the detection method to be used.
There are three types of gels, each requiring a different handling procedure
before they can be added to the blotting sandwich.
Protein
and
agarose
gels set up are arranged together, with
sequencing
gel transfer protocol
following separately.
DNA/RNA:
If these gels were not run in IXTBE, they should be
equilibrated for 10 minutes in this buffer.
Protein Gels
:
After electrophoresis, remove the gel assembly from the apparatus and
remove the spacers.
Open the gel cassette by gently rocking a spatula between the plate, forcing
separation of the plate from the gel. The gel will normally remain affixed to
the bottom plate. Remove the top (notched) plate by slowly lifting it from
the side with the inserted spatula and gradually increasing the angle until
the plate is completely separated from the gel.
If the gel sticks to the top plate in an isolated spot, a stream of water from
a squirt bottle can be sprayed at the spot to aid separation.
Remove the gel from the remaining plate. Tip the plate up side down, and
start one edge, and allow it to roll off into transfer buffer. Alternatively,
place the plate with the gel attached, into transfer buffer.
Incubate the gel in transfer buffer for 15 min. with gentle agitation. If the
gel is on the plate, it will become loose during this step.
Setting Up the Blot
