Specially, in case of tau phosphorylation, the single residue phosphorylation can be detected through western blotting. Is it possible to detect multiple site phosphorylation in one blot? or any other technique is available? If anyone have any idea, i'm requesting to share.
Thanks in advance......
One can at least abstract single site phosphorylation and multiple phosphorylations by just looking at the phosphoshift of the protein using western Blot and a conventional Antibody. But to be specific the above mentioned techniques are needed.
Isoelectric focusing may allow you to detect multiple phosphorylations by gel electrophoresis and Western blot.
Again thanks to all for your important suggestions. I hope it will work for me......
Tau, have an electrophoretic profile with four main bands (Tau 55, 64, 69, 74 kDa).There are a variety of parameters that can be of great help:
In case you want to run WB:
1- I would recommend that you have a PAGE system which separates between 55-74 (possibly 4-12% AA).
2- if you are immuno-precipitate then you can use different ab that recognize different epitopes in one mix.
If you want to run other methods then I would run the 2-D electrophoresis .. I recommend to label the cell extract with CyDyes (Cy3, Cy5 or Cy7) The Control extract with an Dyes and treated / induced with another Dyes and mix download and run 2D. The method can be interpreted a little pricey but it is very effective for detecting modified proteins including phosphorylation in a single blot / image. scanning the gels with different filter to visualize different forms of your protein. . if you are interested in 2D : Check http://www.bloodjournal.org/content/93/11/3900
fig 7 can help.
Good Luck
Agree with Patick and Fraud, according to what protein you are investigating if you do a low density acrylamide gel you can detect a spread of the phosphorylated protein compared to the non-phosphorylated one. If you are looking at different phosphorylation sites you may be able to find specific antibodies for each one that you can use in wb
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