Biomedical Engineering Reference
In-Depth Information
6. Set up the glass plates as instructed by the mini gel electrophoresis
system using the 1.5 mm spacer and short plates and the cast-
ing module.
7. Mix 5 mL Lower Gel Buffer, 5 mL of the 40 % acrylamide/
bis-acrylamide reagent, and 10 mL ddH
2
O in a 50 mL plastic
tube. Add 100
L TEMED to initiate the
cross-linking process. Working quickly, invert and rotate the
tube two or three times to mix gently without introducing air
or frothing (
see
Note 29
). Pour approximately 7.5 mL between
the glass plates until the level is about 15 mm from the top of
the short plate, immediately overlay the surface with water-
saturated butanol, and allow the gel to set. The butanol phase
will seal the gel from air, allowing the cross-linking to proceed.
It should take about 45 min.
8. When the gel is set, remove the butanol and rinse the gel well
with ddH
2
O. Tilt the gel upside down and place it on a piece
of absorbent paper to soak up all the remaining liquid from
between the plates. Prepare 10 mL of 4 % stacking gel by add-
ing 2.5 mL upper gel buffer and 1 mL of acrylamide reagent
to 6.5 mL of ddH
2
O. Mix gently. Add 60
ʼ
L APS and 10
ʼ
L
TEMED and mix and pour to the top of the plates. Immediately
and carefully slide the combs in place until they are fl ush with
the glass plate and allow the gel to set.
9. Remove the combs and set the gel plates into the electropho-
resis module and tank as instructed. Fill the inner chamber to
the top and the outer chamber till it is 5-6 cm from the base
with 1× SDS Running Buffer. Ensure that there are no leaks
between the seal of the glass plates and the rubber gaskets of
the module. Load the gel with 30-50
ʼ
L APS and 6
ʼ
ʼ
g of the protein sam-
L of standard markers.
10. Run the gel at 15 mA for 15 min to run the protein into the
stacking gel; then increase the current to 25 mA and run until
the loading dye reaches the base of the glass plates.
11. Freshly prepare 1 L of 1× transfer buffer and cut a piece of
nitrocellulose membrane 8 cm by 10 cm. Also cut six pieces of
3mm CHR paper to the size of 8 cm by 10 cm. Soak the mem-
brane and CHR paper in 1× transfer buffer.
12. Using a clean tray, add 500 mL of 1× transfer buffer. Submerge
an open transfer cassette (black side down) in the tray. On the
black surface of the transfer cassette, layer a transfer pad, fol-
lowed by two soaked 3mm CHR paper.
13. Insert a plastic separator between the glass plates to release one
glass plate from the gel. Run fl at-tipped forceps between the
spacers and gel to release the gel. Place a soaked 3mm CHR
paper on the gel, invert the plate, gel and paper, and remove
the glass plate from the gel and paper. Place the gel and paper
onto the setup of paper, transfer pad and cassette in the tray.
ples and 5
ʼ
