Pro-Q Diamond Phosphoprotein Gel Stain from Invitrogen (Molecular Probes)

Phosphorylation is an important post translational modification which governs a variety of cellular functions for many proteins. Phosphorylation mainly happens on the serine, tyrosine or threonine residues of proteins. Kinases are enzymes which play a major role in phosphorylating proteins and regulating complex signaling pathways. Since phosphorylation governs such a wide array of cellular mechanisms and functions, it is very vital in understanding the ‘state’ of the protein upon synthesis (phosphorylated/un-phosphorylated). The phosphorylation might happen on multiple residues on a protein.

The use of antibodies has been the standard methodology for detection of phosphorylation status of a protein. These antibodies, which are known as phosphor-specific antibodies, are available in abundance and form an important component of current research technologies. Use of radioactive labels, e.g. ³²P has also been used quite frequently in determining the phosphorylation status of a protein.

The Pro-Q Diamond stain offers a superb alternative to antibodies and radioactive materials for detecting the phosphorylation status of proteins. The stain can be applied directly on the SDS-PAGE gels for identification of phosphor-specific proteins. This is a proprietary fluorescent stain and is compatible with 1D and 2D gels. Detection is easy with visible light-scanning or a visible transilluminator. The emission and excitation mamixa for this stain is approximately 555/580 nm. Depending upon the phosphorylation status of the protein, the stain is sensitive for detection of nanogram levels of proteins. The stain can be reused once or twice without any reduction in the performance or sensitivity. Storage of the used stain in amber colored containers or preventing it from direct exposure to light helps preserve the sensitivity as well as elongating the half life of the stain.

The staining protocol can generally be performed in a time span of 4-5 hours; however, an overnight fixation step definitely helps in increasing the sensitivity of detection, especially for low-abundance phosphorylated proteins. A total protein stain using Coomassie or Sypro following the phospho-protein staining step helps differentiate and identify the specific phopho-proteins of interest. Standard molecular weight markers which are phosphorylated can be purchased with the stain, and is called PeppermintStick Phosphoprotein MW Markers.

I have used the Pro-Q Diamond phospho-protein stain from Invitrogen extensively for my research. My protein is a kinase and I am very interested in figuring the phosphorylation status of the same. I have used the stain both for 1D and 2D gels and it works equally well. For imaging purposes, I have used the Treo scanner from GE and with a high laser (e.g 600 PMT or higher). This has given me very good signal intensity for my phospho-proteins. I have a dedicated Rubbermaid container which I exclusively use for the Pro-Q Diamond staining and the container is cleaned very rigorously every time before use following the recommendations. Overnight fixing of the gel has yielded good results in my case, compared to the shorter time of incubation. One other things worth mentioning is that very delicate handling of the gel is required with minimum contact (even with gloves), which may otherwise lead to black patches on the gel upon imaging. Use of a big spatula wrapped with saran wrap for transfer of the gels does eliminate some of these problems. Overall, it is a very good reagent for detection of phosphorylated proteins: fine tuning might be required on a case-to-case basis for the optimized signal to noise.