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Preparation of denatured protein samples for loading:
The instructions given below are for denatured samples. For Native samples, please
consult a laboratory handbook.
1. Prepare the protein samples for loading. The volume of sample depends on
the capacity of the wells. See well chart in the Appendix.
2. Using a 0.5 ml micro-centrifuge tube or other convenient receptacle, combine
the protein sample and 4 X sample buffer. It is always advisable to use
protein markers in one of the end lanes to indicate sizes of bands. These
should be prepared according to the manufacturer’s instructions.
3. Heat the samples in a water bath or heating block for 2 minutes to denature.
NOTE: heating should be done under a fume hood.
4. Centrifuge the samples in a microcentrifuge for 20 seconds at 12,000 rpm.
The protein samples are now ready to load.
Loading the samples:
1. If desired, fit the previously frozen cooling pack(s) into the tank. The longest side
of the ice pack should be positioned horizontal with the side(s) of the tank and
pressed into the recess. One pack is supplied as standard. Additional packs can
be purchased.
NEVER FIT THESE UNDERNEATH THE MODULE IN THE BOTTOM OF
THE TANK AS THIS WILL PREVENT THE FLOW OF CURRENT
THROUGH THE GEL AND CAUSE SLOW RUNS AND OVER-HEATING.
2. Remove the combs with a gentle rocking motion; rinse the well out with ddi H2O
to eliminate any residue.
3. Transfer the Inner gel module containing cast gels into the main tank in the
correct orientation as indicated +ve on the module aligned with +ve on the tank,
-ve on the module aligned with -ve on the tank.
4. Fill the outer tank with 1 x reservoir buffer. See Appendix for recommended
running buffer solution. Table 6. Shows the volume of buffer required.
