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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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γ-Secretase complex components were specifically captured by GCB pulldown from human frozen brain material. Solubilized γ-secretase prepared from human brain (frontal cortex) was incubated with 200 nM GCB in the presence (+) or the absence (−) of 10 μM L-685,458 and isolated with SA beads. The captured γ-secretase complex was eluted by 10 mM DTT supplement with 0.01% RapiGest and subjected to Western blotting for the indicated γ-secretase subunit. The density of the bands was calculated as a percentage of a standard (input sample) run on the same gel.
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fig06: γ-Secretase complex components were specifically captured by GCB pulldown from human frozen brain material. Solubilized γ-secretase prepared from human brain (frontal cortex) was incubated with 200 nM GCB in the presence (+) or the absence (−) of 10 μM L-685,458 and isolated with SA beads. The captured γ-secretase complex was eluted by 10 mM DTT supplement with 0.01% RapiGest and subjected to Western blotting for the indicated γ-secretase subunit. The density of the bands was calculated as a percentage of a standard (input sample) run on the same gel.

Mentions: Microsomal membranes from human frontal cortex were prepared and extracted as described above, and the optimized conditions were used for pulldown of γ-secretase. The recovery of the γ-secretase components was similar as for rat, and the competition with L-685,458 was efficient (Fig. 6). Thus, we conclude that the present approach is efficient and can be used for purification of γ-secretase also from post-mortem human brain tissue.


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)

γ-Secretase complex components were specifically captured by GCB pulldown from human frozen brain material. Solubilized γ-secretase prepared from human brain (frontal cortex) was incubated with 200 nM GCB in the presence (+) or the absence (−) of 10 μM L-685,458 and isolated with SA beads. The captured γ-secretase complex was eluted by 10 mM DTT supplement with 0.01% RapiGest and subjected to Western blotting for the indicated γ-secretase subunit. The density of the bands was calculated as a percentage of a standard (input sample) run on the same gel.
© Copyright Policy
Related In: Results  -  Collection

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

fig06: γ-Secretase complex components were specifically captured by GCB pulldown from human frozen brain material. Solubilized γ-secretase prepared from human brain (frontal cortex) was incubated with 200 nM GCB in the presence (+) or the absence (−) of 10 μM L-685,458 and isolated with SA beads. The captured γ-secretase complex was eluted by 10 mM DTT supplement with 0.01% RapiGest and subjected to Western blotting for the indicated γ-secretase subunit. The density of the bands was calculated as a percentage of a standard (input sample) run on the same gel.
Mentions: Microsomal membranes from human frontal cortex were prepared and extracted as described above, and the optimized conditions were used for pulldown of γ-secretase. The recovery of the γ-secretase components was similar as for rat, and the competition with L-685,458 was efficient (Fig. 6). Thus, we conclude that the present approach is efficient and can be used for purification of γ-secretase also from post-mortem human brain tissue.

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