Archive for the ‘Antibody’ Category
Advances in Phosphorylation Detection Techniques
We at Novus Biologicals recently extended our antibody catalog with the addition of several new phospho-specific antibodies. Phosphorylation is a key component of all protein pathways, including those associated with diseases like cancer.
Until recently, analysing phosphorylated proteins with antibodies was a tortuous affair. To see if drug treatment or other action had changed a protein’s phosphorylation status, the cells had to be loaded with radioactive phosphate. Following treatment, the protein was removed from the cells and immunoprecipitated with the appropriate antibody. Electrophoresis was then applied to resolve the antibody-antigen complex onto a gel. The only way to speed things up was to take a global perspective, and omit the IP stage.
Either technique produced multiple problems, not least which is that of having to dispose of the resultant radioactive waste afterwards. After all that, the scientist would still be no wiser as to which amino acids had been altered, or to what extent. Some modification sites activate phosphorylation, while others block or deactivate it – knowing which sites are phosphorylated is key to understanding consequent events.
Today, the researcher has access to a wide range of phospho-specific antibodies, gel stains, spectrometers and purification reagents. The simplest approach is a bulk-staining method, using colorimetric or fluorescent phospho gel dyes. The protein is resolved onto a one or two-dimensional polyacrylamide gel, and then stained using dyes specific to both the total and phosphorylated protein.
A much clearer picture is obtained using phospho-specific antibodies, such as CaMKII alpha [phospho Thr286] (calmodulin kinase II phosphorylated at threonine-286) antibody. “Pan” phospho-antibodies also exist, such as EIF4E2 (4G10) antibody, which detects all phosphotyrosine residues. These products can be found on our antibody database.