Phos-tag SDS-PAGE database mTOR

mTOR

Serine/threonine protein kinase which is a central regulator of cellular metabolism, growth and survival in response to hormones, growth factors, nutrients, energy and stress signals. Mw; 289,000

Image

mTORは高分子量(289 kDa) であるため，
3％という低濃度のポリアクリルアミドゲルを用いて解析しています。
3％ではゲルが柔らかすぎて扱いにくいため，0.5％アガロースを加えて補強しています。

HeLa細胞を無血清で培養したのち，
FBSを10％になるように添加して30分後にmTORのリン酸化状態を
Mn²⁺–Phos-tag SDS-PAGEで解析しています。

Reference	Kinoshita, E., Kinoshita-Kikuta, E., Ujihara, H., and Koike, T. Mobility shift detection of phosphorylation on large proteins using a Phos-tag SDS-PAGE gel strengthened with agarose. *Proteomics*, 9, 4098–4101 (2009) doi: 10.1002/pmic.200900020. Kinoshita, E., Kinoshita-Kikuta, E., and Koike, T. Separation and detection of large phosphoproteins using Phos-tag SDS-PAGE.Nature Protocols, 9, 1513–1521 (2009) doi: 10.1038/nprot.2009.154
Composition of gel	Mn²⁺–Phos-tag:　20µM Polyacrylamide: 3%(w/v) + Agarose 0.5%(w/v)
Detection	WB: anti-mTOR anti-pmTOR(pS2448) anti-pmTOR(pS2481)

Reference

Kinoshita, E., Kinoshita-Kikuta, E., Ujihara, H., and Koike, T. Mobility shift detection of phosphorylation on large proteins using a Phos-tag SDS-PAGE gel strengthened with agarose. Proteomics, 9, 4098–4101 (2009)

doi: 10.1002/pmic.200900020.

Kinoshita, E., Kinoshita-Kikuta, E., and Koike, T. Separation and detection of large phosphoproteins using Phos-tag SDS-PAGE.Nature Protocols, 9, 1513–1521 (2009)

doi: 10.1038/nprot.2009.154

Composition of gel

Mn²⁺–Phos-tag:　20µM

Polyacrylamide: 3%(w/v)
+
Agarose 0.5%(w/v)

Detection

WB:
anti-mTOR

anti-pmTOR(pS2448)

anti-pmTOR(pS2481)