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Affinity pulldown of γ-secretase and associated proteins from human and rat brain.

Teranishi Y, Hur JY, Welander H, Frånberg J, Aoki M, Winblad B, Frykman S, Tjernberg LO - J. Cell. Mol. Med. (2010)

Bottom Line: After pulldown using streptavidin beads, bound proteins were eluted under reducing conditions and digested by trypsin.Interestingly, TMP21 and the PS associated protein syntaxin1 were associated to γ-secretase in rat brain.We suggest that the present method can be used for further studies on the composition of the γ-secretase complex.

View Article: PubMed Central - PubMed

Affiliation: The Karolinska Institutet (KI) Dainippon Sumitomo Pharma Alzheimer Center (KASPAC), KI-Alzheimer's Disease Research Center, Department of Neurobiology, Care Sciences and Society, Karolinska Institutet, Novum, Huddinge, Sweden.

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The structure of biotinylated γ-secretase inhibitor and the inhibitory potency for Aβ40 production. (A) GCB is designed with a long hydrophilic linker, a disulfide bond and a biotin group. The distance between the inhibitor part and the biotin moiety is determined by 3D ChemDraw (CambridgeSoft, Cambridge, MA, USA). (B) Dose inhibition curves of GCB and L-685,458. Inhibitory potencies were measured by the in vitro generation of Aβ40 in the endogenous substrate assay using membrane fraction from BD8-PS1cells.
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fig01: The structure of biotinylated γ-secretase inhibitor and the inhibitory potency for Aβ40 production. (A) GCB is designed with a long hydrophilic linker, a disulfide bond and a biotin group. The distance between the inhibitor part and the biotin moiety is determined by 3D ChemDraw (CambridgeSoft, Cambridge, MA, USA). (B) Dose inhibition curves of GCB and L-685,458. Inhibitory potencies were measured by the in vitro generation of Aβ40 in the endogenous substrate assay using membrane fraction from BD8-PS1cells.

Mentions: The molecule was designed for efficient pulldown of γ-secretase. We coupled the commonly used inhibitor L-685,458 via a long hydrophilic linker to a cleavable biotin group (GCB) (Fig. 1A). The inhibitor and the biotin group are around 70 Å apart (as estimated by 3D ChemDraw), which is sufficient for concurrent binding of γ-secretase and SA. The inhibitory potency was tested by quantifying Aβ production from microsomal membranes prepared from BD8-PS1 cells by ELISA, and GCB showed similar IC50 as L-685,458 (Fig. 1B). Thus, we conclude that the linker-biotin part has only a minor effect on affinity, and that GCB may be suitable for pulldown of γ-secretase.


Affinity pulldown of γ-secretase and associated proteins from human and rat brain.

Teranishi Y, Hur JY, Welander H, Frånberg J, Aoki M, Winblad B, Frykman S, Tjernberg LO - J. Cell. Mol. Med. (2010)

The structure of biotinylated γ-secretase inhibitor and the inhibitory potency for Aβ40 production. (A) GCB is designed with a long hydrophilic linker, a disulfide bond and a biotin group. The distance between the inhibitor part and the biotin moiety is determined by 3D ChemDraw (CambridgeSoft, Cambridge, MA, USA). (B) Dose inhibition curves of GCB and L-685,458. Inhibitory potencies were measured by the in vitro generation of Aβ40 in the endogenous substrate assay using membrane fraction from BD8-PS1cells.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4373488&req=5

fig01: The structure of biotinylated γ-secretase inhibitor and the inhibitory potency for Aβ40 production. (A) GCB is designed with a long hydrophilic linker, a disulfide bond and a biotin group. The distance between the inhibitor part and the biotin moiety is determined by 3D ChemDraw (CambridgeSoft, Cambridge, MA, USA). (B) Dose inhibition curves of GCB and L-685,458. Inhibitory potencies were measured by the in vitro generation of Aβ40 in the endogenous substrate assay using membrane fraction from BD8-PS1cells.
Mentions: The molecule was designed for efficient pulldown of γ-secretase. We coupled the commonly used inhibitor L-685,458 via a long hydrophilic linker to a cleavable biotin group (GCB) (Fig. 1A). The inhibitor and the biotin group are around 70 Å apart (as estimated by 3D ChemDraw), which is sufficient for concurrent binding of γ-secretase and SA. The inhibitory potency was tested by quantifying Aβ production from microsomal membranes prepared from BD8-PS1 cells by ELISA, and GCB showed similar IC50 as L-685,458 (Fig. 1B). Thus, we conclude that the linker-biotin part has only a minor effect on affinity, and that GCB may be suitable for pulldown of γ-secretase.

Bottom Line: After pulldown using streptavidin beads, bound proteins were eluted under reducing conditions and digested by trypsin.Interestingly, TMP21 and the PS associated protein syntaxin1 were associated to γ-secretase in rat brain.We suggest that the present method can be used for further studies on the composition of the γ-secretase complex.

View Article: PubMed Central - PubMed

Affiliation: The Karolinska Institutet (KI) Dainippon Sumitomo Pharma Alzheimer Center (KASPAC), KI-Alzheimer's Disease Research Center, Department of Neurobiology, Care Sciences and Society, Karolinska Institutet, Novum, Huddinge, Sweden.

Show MeSH
Related in: MedlinePlus