Biology Reference
In-Depth Information
11. Group the individual images together into their respective
classes (e.g. diet 1 and diet 2, genotype 1 and genotype 2,
control and treated).
12. Review each of the detected spots. There are certain criteria to
be met in deciding which gel spots truly refl ect a change in
expression levels between any two conditions (diet, genotype,
etc.). Tag all of the spots where the ANOVA p-value is
<0.05 and fold change is greater than an appropriate level,
for example, 2.
13. From the “Progenesis Stats” icon, additionally tag those spots
where statistical power is greater than 80% (the generally
accepted threshold level). It is thus possible to scrutinise and
reduce the list of detected proteins and by selecting only
those peaks that have, for example,
p
< 0.05, fold change >2,
and power >0.8.
14. Visually examine these signifi cant spots on the gel image to
ensure that they can realistically be picked—and are not due
to artefacts such as streaking.
Once the protein spots of interest have been identifi ed they can be
excised robotically or stained, visualised and excised manually. It is
possible to excise from either one of the gels used in the DIGE
experiment, or from a preparative gel that has been prepared and
run simultaneously but without the use of fl uorescent dyes, and
with a much higher protein loading.
In this example, protein spots of interest were manually excised
from the analytical gels. The staining method used has high sensi-
tivity similar to that of silver staining—yet it is compatible with
mass spectrometry (
4
). It is advisable to pick the spots from a gel
where the protein spots of interest have a high intensity—for this
reason it may be necessary to stain more than one gel:
3.7. Visualisation
and Picking of
Proteins of Interest
1.
Clean and dry the exterior of the gel cassette and lay it onto
lint-free paper with the smaller of the two glass plates in con-
tact with the paper.
2.
Prise off the top glass plate carefully to avoid damaging the gel.
Remove the IPG strip and agarose, and place the gel assembly
gel-side up in a clean staining tray.
3. Cover the gel with fi xing solution (see Subheading
2.2.7
) and
place foil over the staining tray. Fix the gel overnight on a shaker.
4.
Pour off the fi xative and rinse the gel 3 times with deionised
water.
5.
To prepare 1 L of colloidal Coomassie stain, dissolve 100 g of
ammonium sulphate in 500 mL of deionised water on a stirrer,
and add 1.2 g of G250 Coomassie blue and 118 mL of phos-
phoric acid. When it has all dissolved, make the solution up to
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