11
Stacking Gel:
Solution
Volume
Distilled Water
4.2ml
30 % Stock Acrylamide Solution
0.65ml
4 X Stacking Gel Tris Solution
1.6ml
Add 67µl of 10 % Ammonium Persulphate and 6.7µl TEMED before pouring.
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
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 the samples.
4.
Centrifuge the samples in a micro-centrifuge for 20 seconds at 12,000
rpm. The protein samples are now ready to load.
Gel Pouring
Casting a gel with stacking layer
1.
Place a comb into the gel cassette assembly with any gel and mark
the glass plate below the comb teeth. This is the reference level to
which the resolving gel should be poured.
2.
Prepare the resolving gel solution. Mix well and avoid generating air
bubbles.
