Robert Amezquita
PhD Scholar
Yale

Sonam, MA, US

Is there any method to detect whether a protein is at all active (phosphorylated) using a tool such as an antibody that targets all or multiple phosphorylation sites?

Robert Amezquita
HHMI Gilliam Fellow,
PhD Scholar,
Immunology,
Yale
Yes, there are methods to detect activated proteins, or in other words, proteins that have undergone posttranslational modifications. Such modifications include phosphorylation, but also ubiquitination (a signal for degradation), methylation, acetylation, and many others.

The most popular detection tool for these proteins is exactly what you mentioned in your question: an antibody, which can be used to detect any and all of these posttranslational modifications. An antibody is an amazing molecular tool that allows for the detection of any specific motif. The tricky part of this technique is generating the right antibody for your target. Many companies sell a wide range of antibodies with different target protein specificities to suit your needs. However, if for some reason you cannot find the right antibody, you may need to generate your own  (here is a step-by-step tutorial).

The next step is being able to detect the binding of the antibody to your target protein (modification). You can do this by conjugating (combining) the antibody with a molecular marker to make it detectable. The most common, and simplest, method for antibody detection is by conjugating it with a fluorophore. The fluorophore is detectable when exposed to light of a particular wavelength, re-emitting light at a different wavelength. Other detection methods include enzymatic techniques such as in the ELISA (enzyme-inked immunosorbent assay), which uses an enzyme conjugated to an antibody to turn a colorless substrate into something detectable, as shown here.

09/17/12 15:48