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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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Syntaxin1 and TMP21 are GSAPs in rat brain. Solubilized γ-secretase prepared from rat brain were incubated with 200 nM GCB in the presence (+) or the absence (−) of 10 μM L-685,458 and isolated with SA beads. (A) The captured samples from rat brain in the presence (blue line) or the absence (red line) of 10 μM L-685,458 were digested with trypsin and analysed by LC-MS/MS. Extracted ion chromatogram for m/z 1015.5 corresponding to the tryptic peptide RLEDLSESIVNDFAYMK from TMP21. (B) Extracted ion chromatogram for m/z 816.5 corresponding to the tryptic peptide SIEQSIEQEEGLNR from syntaxin1. (C, D) The captured samples were eluted with SDS sample buffer and subjected to Western blotting for TMP21 (C) and syntaxin1 (D).
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fig04: Syntaxin1 and TMP21 are GSAPs in rat brain. Solubilized γ-secretase prepared from rat brain were incubated with 200 nM GCB in the presence (+) or the absence (−) of 10 μM L-685,458 and isolated with SA beads. (A) The captured samples from rat brain in the presence (blue line) or the absence (red line) of 10 μM L-685,458 were digested with trypsin and analysed by LC-MS/MS. Extracted ion chromatogram for m/z 1015.5 corresponding to the tryptic peptide RLEDLSESIVNDFAYMK from TMP21. (B) Extracted ion chromatogram for m/z 816.5 corresponding to the tryptic peptide SIEQSIEQEEGLNR from syntaxin1. (C, D) The captured samples were eluted with SDS sample buffer and subjected to Western blotting for TMP21 (C) and syntaxin1 (D).

Mentions: In the samples analysed above, TMP21 and syntaxin1 were identified with high scores (TMP21 = 416 and syntaxin1 = 119), clearly above the significance level of 40. The selected ion chromatograms corresponding to the identified peptides showed competition by L-685,458 (Fig. 4A and B). The competition of these proteins was validated by Western blotting (Fig. 4C and D). Hence, we suggest that these proteins can interact with γ-secretase in rat brain.


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)

Syntaxin1 and TMP21 are GSAPs in rat brain. Solubilized γ-secretase prepared from rat brain were incubated with 200 nM GCB in the presence (+) or the absence (−) of 10 μM L-685,458 and isolated with SA beads. (A) The captured samples from rat brain in the presence (blue line) or the absence (red line) of 10 μM L-685,458 were digested with trypsin and analysed by LC-MS/MS. Extracted ion chromatogram for m/z 1015.5 corresponding to the tryptic peptide RLEDLSESIVNDFAYMK from TMP21. (B) Extracted ion chromatogram for m/z 816.5 corresponding to the tryptic peptide SIEQSIEQEEGLNR from syntaxin1. (C, D) The captured samples were eluted with SDS sample buffer and subjected to Western blotting for TMP21 (C) and syntaxin1 (D).
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fig04: Syntaxin1 and TMP21 are GSAPs in rat brain. Solubilized γ-secretase prepared from rat brain were incubated with 200 nM GCB in the presence (+) or the absence (−) of 10 μM L-685,458 and isolated with SA beads. (A) The captured samples from rat brain in the presence (blue line) or the absence (red line) of 10 μM L-685,458 were digested with trypsin and analysed by LC-MS/MS. Extracted ion chromatogram for m/z 1015.5 corresponding to the tryptic peptide RLEDLSESIVNDFAYMK from TMP21. (B) Extracted ion chromatogram for m/z 816.5 corresponding to the tryptic peptide SIEQSIEQEEGLNR from syntaxin1. (C, D) The captured samples were eluted with SDS sample buffer and subjected to Western blotting for TMP21 (C) and syntaxin1 (D).
Mentions: In the samples analysed above, TMP21 and syntaxin1 were identified with high scores (TMP21 = 416 and syntaxin1 = 119), clearly above the significance level of 40. The selected ion chromatograms corresponding to the identified peptides showed competition by L-685,458 (Fig. 4A and B). The competition of these proteins was validated by Western blotting (Fig. 4C and D). Hence, we suggest that these proteins can interact with γ-secretase in rat brain.

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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Related in: MedlinePlus