Phosphorylation-Specific Antibody Introduction

Protein phosphorylation modification is one of the most common forms of post translational modifications. It plays an important role in regulating signal transduction, gene expression, the cell cycle, and many other physiological processes. Phosphorylation-specific antibodies are an important tool for studying protein phosphorylation. Based on a well-established antibody discovery platform, Creative Biolabs can produce high-affinity phosphorylation-specific antibodies for you.

Phosphorylation Modification

Protein phosphorylation modification is the most important form of covalent modification in protein post-translational modifications. It is also the most important form of regulatory modification in prokaryotes and eukaryotes. Protein phosphorylation modifications are widely found in microorganisms, plants, and animals. At least 30% of proteins in living organisms are modified by phosphorylation. In general, the amino acid residues that undergo phosphorylation will differ from species to species. In eukaryotes, phosphorylation occurs mainly on residues such as serine, threonine, and tyrosine. In contrast, in bacteria, proteins are phosphorylated mainly through residues such as aspartate, glutamate, and histidine. Some proteins can be phosphorylated in both prokaryotes and eukaryotes, and their phosphorylation sites are usually arginine, lysine, and cysteine residues.

Phosphorylation-Specific Antibody

Protein PTM research cannot be done without the help of PTM-specific antibodies. However, the development of PTM specific antibodies is difficult, and the quality control of the finished product is also difficult. This has greatly hindered the progress of PTM research. The same is true for phospho-specific antibodies. Phospho-specific antibodies are antibodies that can be used to detect changes in the phosphorylation level of a protein when the cell is stimulated. With the exception of common phosphorylated proteins for which commercially available antibodies are available, most phosphorylated protein antibodies require custom development when used.

Phosphorylation-specific antibodies are prepared primarily against phosphorylation sites and can specifically recognize phosphorylated amino acid sites. Phosphorylated protein antibodies can detect the level of protein phosphorylation and perform qualitative and quantitative analyses of phosphorylated proteins. Phosphorylated protein antibodies have a wide range of applications in immunology, biochemistry, oncology, proteomics, and especially in the life sciences, such as cell signaling. apoptosis, and cancer research.

Tips for Using Phosphorylation-Specific Antibodies

The phosphorylation of proteins is reversible. Therefore, if care is not taken during the experiment, it may lead to dephosphorylation of the phosphoprotein, which will definitely result in a failed experiment. Therefore, extra care needs to be taken when using phosphorylation-specific antibodies to detect phosphorylated proteins. We will share an example of commonly used western blotting.

First, during protein extraction, the action should be fast, the sample should be fresh, preferably on ice, and the operation time should be as short as possible. Immediately after protein quantification is completed, the extracted phosphorylated proteins are denatured and stored at -80°C in batches to ensure minimal degradation and to avoid degradation of the phosphate group due to repeated lyophilization. To prevent the dephosphorylation of phosphorylated proteins, we can add phosphatase inhibitors to the protein extract, loading buffer, and transfer buffer. Next is the blocking process. Prolonged blocking can mask antigenic epitopes and prevent antibody binding. Therefore, too long a blocking time needs to be avoided during the operation. And the time to wash the blot should not be too long; a long washing time may lead to the antibody being eluted, and thus the signal is weakened. Finally, a good phosphorylated protein antibody is a key factor for the success of the experiment. Antibody dilutions and procedures should be optimized according to specific experimental results.

Creative Biolabs has a wealth of knowledge about antibody discovery. We would be happy to discuss with you our knowledge and experience in phospho-specific antibody development.

All listed services and products are For Research Use Only. Do Not use in any diagnostic or therapeutic applications.

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