New way to measure isoelectric point of proteins

September 10, 2012 § Leave a comment

Nanoparticle-based system for measuring protein pI. Image provided by Harri Härmä .

Need to find out your favorite protein’s isoelectric point but can’t bear the thought of running yet another messy and time-consuming gel? This newsflash may be interesting to you: Researchers have come up with a new method of estimating a protein’s pI that is fast and simple — and doesn’t involve separating proteins in a pH gradient.

A pI, the pH at which a molecule doesn’t have a net electric charge, is usually measured by isoelectric focusing in either a gel or capillary format. But, as Harri Härmä at the University of Turku in Finland explains, the methods demand high concentrations of sample, special instrumentation and multiple assay components, solutions and accessories. To top it all off, “they are laborious and time consuming,” he says.

Härmä and his team developed a luminescence assay using nanoparticles. As they described in their Analytical Chemistry paper, they labeled nanoparticles with a europium ion chelator.  When a protein is at a pH higher than its pI, it doesn’t adsorb to the nanoparticle. This lack of adsorption makes the europium ion dissociate, and the nanoparticle does not give off a strong luminescent signal.

However, when the protein is at a pH lower than its pI, it adsorbs to the nanoparticle and stops the europium ion from dissociating. The nanoparticle gives off a strong luminescent signal.

The experiment can be run in a microtiter plate that has the pH gradient over the desired range. All the necessary reagents are already prepared and ready to go in the wells in a dry form. 

All a researcher has to do is mix in the protein of interest with the appropriate buffer and the nanoparticles. By reading off luminescent signals with a standard plate reader, a researcher can get a quick estimate of a protein’s pI.

The investigators say that their method is over 1,000 times more sensitive than conventional methods and requires less sample. Härmä and his team confirmed that their method worked with four proteins – BSA, carbonic anhydrase, γ-globulin and trypsinogen – with pIs ranging from 5 to 9. They also made sure the assay was not susceptible to interfering chemicals, such as non-ionic surfactants and ethanol.

“New means for the detection of protein isoelectric point is of a great interest to the scientific community,” says Härmä. He adds that the same technology platform can be adapted easilyto measure other biochemical parameters, such as concentration and size, and to check for quality of protein preparations.

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