Biomedical Engineering Reference
In-Depth Information
Expel the excess water with a photo roller. Place a second glass plate
bearing the U-frame (for 0.5 mm distance) on top and clamp the
cassette together. Cool this sandwich for 30 min in a refrigerator.
Give 8.1 ml of the acidic solution (mixture as given in Table 2.10
without Soln. C) into the “ds” chamber of the gradient mixer, open
the connecting valve for a moment to let air out, then pour 8.1 ml of
the basic solution (mixture as given in Table 2.10 without Soln. C)
into the “ls” chamber. Add Soln. C to start polymerization, switch
on the magnetic stirrer, and open both the valves.
When the cassette is filled after 2 - 3 min, allow the gradient to
equilibrate for about 15 min at RT. The polymerization is completed
for 1 h at 50
◦
C or overnight at RT.
Remove the IPG gel from the cassette and wash five to six
timesinaglasstrayfor10min each with 500 ml deionized water.
Equilibrate the gel in 2% glycerol (w/v) for 30 min at RT, and then
dry it overnight in a dust-free cabinet.
Cover the dry gel with a protective plastic foil, cut it into 5 mm
strips and store airtight at −20
◦
C (strips are stable for several
months).
Pour 300 - 400
µ
l of rehydration buffer D
11
intoasmalltray.
Rehydration of
Immobiline gels
Peel off the protective foil and insert the IPG gel face down into
the tray. Cover gel and rehydration buffer with 1 - 2 ml silicone oil,
close the lid of the tray, and allow swelling overnight at RT by gentle
agitation.
Apply some drops of kerosene on the cooling block of the hor-
Isoelectric focusing
izontal electrophoresis apparatus and apply the IPG strips with
the acidic side to the anode. Make 1 cm broad strips from 2 mm
thick filter paper. Wet the strips with deionized water (no ultra-
pure water!) and place them on top of the IPG strips as well
as at the anodic (acidic) and the cathodic (basic) ends of the
strip(s).
Use a silicone rubber frame (sample applicator) or a piece of
filter paper (2
5 mm) for sample application about 5 mm from
the anode or cathode and apply sample (concentration should not
exceed 10 mg
×
/
ml), which is dissolved in rehydration buffer.
Place the electrodes and press them gently on the IEF electrode
strips. Close the lid of the electrophoresis apparatus and switch on
voltage. For 180 mm IPG strips the following conditions should be
used (Table 2.11):
Temperature 20
◦
C, maximal current 0.05 mA
/
strip, maximal
/
voltage 3500 V, maximal power 0.2 W
strip. For orientation the
following schedule is used:
Remove the strips using tweezers after focusing. If the second
rundoesnotfollowimmediately,thestripsmaybefrozenat−70
◦
C.
/
11
Rehydration and sample application may be combined. Up to 5 mg
ml
of sample can be added to the buffer D, if the M
r
of the proteins are not
to high or their pI to extreme.
