Biology Reference
In-Depth Information
Fig. 4. Examples of PTMs analyzed by 2D DIGE. (
a
) Ubiquitination, deubiquitinated proteins are indicated by the
arrows
.
(
b
) Phosphorylation, the
inserts
show how the dephosphorylated spots migrate differentially in the pI axis.
4. Notes
1. Concentrated HCl (12N) is fi rst used to make large adjust-
ments to the pH until close to the desired range. A lower con-
centration, such as 1N, is then used to make fi ne adjustments
until the desired pH is reached.
2. In this example, we use calf intestinal alkaline phosphatase
which is suggested by the manufacturer to remove phosphory-
lated serine, threonine, and tyrosine amino acid residues.
However, not all sources/isoforms of alkaline phosphatase
remove phosphates from these amino acids with equal effi -
ciency. In addition, lambda alkaline phosphatase also removes
phosphates from histidine residues. Therefore, it is possible to
identify a residue-specifi c or complete phosphoproteome by
using specifi c phosphatases or a cocktail of phosphatases cover-
ing all possible phosphorylated residues.
3. Here, we spin the samples in a speed vacuum dryer at 4,000 rpm,
at 37°C, until dry. The lid of the speed vacuum dryer is trans-
parent, so it is covered with aluminum foil.
4. The IPG buffer pH should match the pH of the strip. Use the
highest grade of DTT available and store at 4°C.
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