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. 2010 Jul:Chapter 18:Unit 18.16.
doi: 10.1002/0471142727.mb1816s91.

Analysis of protein tyrosine phosphatases and substrates

Fatih Mercan  1 , Anton M Bennett
Affiliations

Analysis of protein tyrosine phosphatases and substrates

Fatih Mercan et al. Curr Protoc Mol Biol. 2010 Jul.

Abstract

Protein tyrosine phosphorylation is a reversible post-translational modification that is essential for life in eukaryotic cells. The combinatorial action of both protein tyrosine kinases and protein tyrosine phosphatases (PTPs) determines the net level of cellular tyrosine phosphorylation. This unit discusses methods to determine the level of protein tyrosine phosphatase activity and methods for discovering novel substrates for protein tyrosine phosphatases.

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Figures

Figure 18.16.1
Figure 18.16.1
pNPP chemistry.
Figure 18.16.2
Figure 18.16.2
An example 96-well plate layout for the Malachite Green assay.
Figure 18.16.3
Figure 18.16.3
An example standard curve for the Malachite Green assay.
Figure 18.16.4
Figure 18.16.4
A hypothetical data set for the Malachite Green assay.
Figure 18.16.5
Figure 18.16.5
Schematic representation of an in-gel phosphatase assay. Cell lysates are prepared and resolved as described in the text. Lane 1: unstimulated, and lane 2: stimulated cells. Immunoprecipitated phosphatase complex from, lane 3: unstimulated, and lane 4: stimulated cells. The gel is processed as described in the text and the “white” areas represent regions in which there is phosphatase activity towards the in-gel substrate. The arrow indicates the position of the immunoprecipitated phosphatase at 69 kD, which exhibits increased phosphatase activity following stimulation. In addition, upon stimulation there is an associated phosphatase activity (~150 kDa).
Figure 18.16.6
Figure 18.16.6
Flow chart of a substrate-trapping experiment.
Figure 18.16.7
Figure 18.16.7
Serum-deprived WI38 cells were pervanadate-treated and lysates were subjected to affinity precipitation with the indicated PTP wild type and substrate-trapping GST fusion proteins. Affinity purified protein complexes were detected by immunoblotting with anti-phosphotyrosine (pTyr) antibodies. The arrow at right indicates the substrate-trapped tyrosyl phosphorylated proteins p100 and p80. Modified from Kolli et al. (2004).
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References

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