Protein Phosphorylation Detection by GST in Vitro

The In Vitro GST (Glutathione S-Transferase) Purification Protein Phosphorylation Detection is an experimental method for studying the phosphorylation status of proteins. This method combines the purification technology of GST fusion proteins with the detection technology of phosphorylation status, used to analyze the phosphorylation level of specific proteins or protein domains. Through this method, researchers can study the phosphorylation status of proteins under specific signal pathway activation or inhibition conditions in vitro.

 

Analysis Workflow

1. Protein Expression and Purification

(1) Express the GST fusion protein in an appropriate host cell (usually E. coli).

(2) Perform affinity chromatography purification using a glutathione agarose column.

(3) Check the purity and integrity of the purified protein, usually through SDS-PAGE gel electrophoresis.

 

2. Phosphorylated Protein Detection

Phosphorylation status detection is usually performed through the following methods:

 

(1) Western Blot

Perform Western Blot analysis using antibodies against phosphorylated proteins. This requires specific antibodies against phosphorylated sites, or antibodies against phosphorylated tyrosine, serine, or threonine.

 

(2) Mass Spectrometry Analysis

Enzymatically digest the purified protein, then perform mass spectrometry analysis to identify phosphorylated amino acid residues.

 

(3) Phosphorylation-Specific Staining

Using phosphorylation-specific staining reagents such as Pro-Q Diamond, phosphorylated proteins can be directly detected on the gel.

 

3. Validation and Quantification

(1) If using Western Blot, the specificity of the signal can be verified by a control group (such as samples treated with phosphatase).

(2) For mass spectrometry data, use corresponding software for data analysis and quantification of phosphorylation sites.

(3) Phosphorylation-specific staining usually provides quantitative information and can be analyzed by image analysis software.

 

4. Functional Validation(optional)

If possible, further validate the effect of phosphorylation on protein function through in vitro or in vivo experiments.

 

Through these methods, the phosphorylation of in vitro purified GST fusion proteins can be detected and analyzed, as well as the specific amino acid residues where phosphorylation occurs.