Biology Reference
In-Depth Information
2.
Add a small amount (ca. 15-20 mg) of the powdered tissue to
an Eppendorf tube and immediately add 1 mL of lysis buffer.
Vortex and then sonicate the Eppendorf tube until the pow-
dered tissue is dissolved.
3.
Centrifuge the tube at 6°C and 12,000 ×
g
for 10 min to pellet
the cell debris. Pipette off the supernatant, aliquot and store
(as required) at −80°C.
4.
Estimate the concentration of protein in the supernatant using
a modifi cation of the Bio-Rad Protein Assay, which is based
on the Bradford method (
3
), by diluting the colour reagent
5 times with water, then adding 20 mL of each sample or
standard to 1.6 mL of the diluted colour reagent in a semi-
microcuvette (see Note 2).
3.2. Protein Labelling
As two samples can be run per gel, the minimum number of gels
will be half of the total number of samples to be analysed. Thus, if
there are 20 samples to be run, it will be necessary to run ten gels.
It is useful to prepare a randomised gel plan in advance, showing
which samples will be labelled with the Cy3 and the Cy5 dyes,
respectively, and how these will be combined on the gels. Table
1
shows such a plan for four different combinations of genotypes
and diets and fi ve biological replicates for each combination. Each
gel is typically loaded with a total of 300 mg of protein: 100 mg
from each of the two samples and a further 100 mg from the pooled
internal standard, which is labelled with the Cy2 dye:
Table 1
Example gel plan showing the labelling scheme for two
genotypes (Gen1 and Gen2), two diets (Diet1 and Diet2)
and fi ve biological replicates of each possible combination
of genotype and diet
Gel no.
Cy2
Cy5
Cy3
1
Pool
Gen1/Diet1-1
Gen1/Diet2-1
2
Pool
Gen1/Diet2-2
Gen2/Diet2-1
3
Pool
Gen2/Diet1-1
Gen1/Diet1-2
4
Pool
Gen2/Diet2-2
Gen2/Diet1-2
5
Pool
Gen1/Diet1-3
Gen2/Diet1-3
6
Pool
Gen2/Diet2-3
Gen1/Diet2-3
7
Pool
Gen2/Diet2-4
Gen1/Diet1-4
8
Pool
Gen1/Diet2-4
Gen2/Diet1-4
9
Pool
Gen2/Diet1-5
Gen2/Diet2-5
10
Pool
Gen1/Diet1-5
Gen1/Diet2-5
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