Summary
There are so many questions about protein phosphorylation. The way for detecting the intracellular protein phosphorylation without phospho-specific antibodies is the most popular question in this field. This post will introduce the methods for detecting the intracellular protein phosphorylation.
Body
If there are no phosphor-specific antibodies, how can we detect the intracellular protein phosphorylation? Here is the way.
The most popular method for this is the 32P radioactive labeling. In order to let the 32P get into protein, you can handle cells with the radioactive 32P-labeled ATP. 2 hours later, you can extract protein after cracking cells. And then you can do the 2D electrophoresis to detect the Phosphoprotein. However, the radiation has its own disadvantages. Because some proteins can be added with a little radioactive phosphate, they are not so easy to be detected. Proteins, which have been detected, can be identified with tandem mass spectrometry sequencing. If it is necessary to analyze the place of phosphorylation, you need to gather peptides with radioactivity in order to do the sequence analysis. Of course, this refers to the separation of the phosphoprotein.
The other method for detecting the Phosphoprotein is Chromatography. The common way for analyzing phosphoprotein is to digest the Phosphoprotein with trypsin to get the specific polypeptide fragments, and then it is separated by reversed phase columns combined with electrospray ionization mass spectrometry.
Immunoprecipitation
Currently, antibodies, which are used to enrich the phosphorylated tyrosine, serine and threonine protein, have been commercialized. But only the anti-phosphotyrosine antibody is very efficient in detecting phosphorylation protein.
Chemical modification
The phosphoprotein can be also gained by using chemical modification of phosphate. There are two methods of chemical modification---β2 elimination reactions and condensation reaction of carbodiimide.
Mass Spectrometry
MALDI2MS analysis has been successfully used to identify non-phosphorylated proteins. This analysis is mainly relying on obtaining the peptide mass fingerprinting to obtain sequence information of the protein. In recent years, tandem mass spectrometry combined with MALDI2MS is also applied to analyze the phosphoprotein. It cannot only detect the phosphoprotein, but also determine the place of phosphoprotein.
We have mentioned so many methods for detecting the phosphoprotein. However, there is not an independent way to achieve this goal. For this purpose, many materials should be gathered.