Phos-tag™ PAGE GUIDEBOOK

Protocol

[ Ⅰ ] Mn2+-Phos-tag™ SDS-PAGE

Note:Always prepare the gel just before use

*1) For the MnCl2 solution, two times the Phos-tag™ concentration(molar ratio) is added.

*2) The normally used concentration can be used for TEMED and Sol.G. The above added amounts are examples.

*3) Add the agarose after distilled water has been added and thoroughly dissolved in a microwave oven and it is still hot.

*4) If necessary, preheat the pipette tip and gel preparation table to 40 - 45℃.

[ Ⅱ ] Tips for Western blotting of Phos-tag™ SDS-PAGE gels -important-

After electrophoresis, an additional procedure, i.e., elimination of the manganese ion (Mn2+) from the gel using chelating agent (EDTA), is necessary before electroblotting. This procedure increases the transfer efficiency of the phosphorylated and non-phosphorylated proteins onto a PVDF membrane.

  • 1)Just after electrophoresis, the gel is soaked in a general transfer buffer containing 1〜10 mmol/L EDTA for a minimum of 10 minutes with gentle agitation. (for 10 minutes x 1〜3 times).
    ※Change the temperature and treatment time with transfer EDTA-buffer according to the gel thickness, etc.(eg: 1.5 mm thick: 20 minute treatment x twice).
    ※ Besides transfer buffer, 1 x Running buffer can be also used.

  • 2)Next, the gel is soaked in a general transfer buffer without EDTA for 10 minutes with gentle agitation(for 10 min. x 1 time).
    ※a wet-tank method is strongly recommended for effective protein transfer from the Mn2+-Phos-tag™ acrylamide gel to the PVDF membrane. (The semi-dry method can also be used.)
    ※ The blotting conditions, such as time and temperature, must be optimized for your phosphorylated target protein in the Phos-tag™ gel

Phos-tag™ SDS-PAGE

[ Ⅲ ] Tips for Mass Spectrometry of Phos-tag™ SDS-PAGE

No special procedures such as EDTA treatment are necessary.