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Open Forum on Methodology

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Shift assay

19 June 2001

Eusebio Perdiguero

Hello,

I guess that you are speaking about mammalian cells. I am working in Xenopus oocyte maturation and I am always getting only one band (Cell Signalling antibody, 1:500 or 1:1000 in 1 % milk RT overnight). But appart of using this dual-phosphorylation antibody I use also a electrophoretic shift assay using a normal polyclonal antibody raised against the C-terminus of Xenopus ERK1. If you have a control where the protein is not phosphorylated you can see a very nice up-shift of the activated protein (I normally use 15% Anderson gels, with standard Laemmli gels it is not detectable).

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