Use of Fluorescent Protein in Studying Protein Half-Life

How long a protein remains in cell and at what equilibrium level depends on several factors: 1) how fast it is translated; 2) how fast it is degraded; 3) how much dilution by cell division affects its balance. A good method for tracking protein degradation requires live cell measurement methods that show high resolution because the changes may be small and gradual; and that do not interfere with cellular processes. One simple method was recently described in Science by Eden et al. that relies on bleaching fluorescent protein (FP) tagged to cellular protein of interest.

To track protein half-life, only a small fraction of FP is bleached with a pulse of light that would irreversibly damage the chromophore of the FP. This treatment, called bleach chase, would produce a population of proteins that are non-fluorescent and cannot be replenished. By comparing the fluorescence of this population and the control, unbleached population, it is possible to determine the half-life of the fused proteins using equation T1/2=ln(2)/a, where a is the slope of decay of the difference between bleached and unbleached protein fluorescence on a semilogarithmic plot. (This part is recited from AlleleNews)

Conversely, instead of photobleaching a FP to create a protein population, a fluorescent signal can be created and chased by photoactivating a photoactivable FP that is fused to a cellular protein under study. Plachta et al. published in a recent issue of Nature Cell Biology that by following the half-life, or kinetics of pluripotency-related transcription factor Oct4, cell fates are predicted in early embryo development.

In fact, there is a third method, perhaps soon to be published, that a photoconvertible FP can be used for tracking fusion protein half life. By using a photoconvertible FP, such as mClavGR (already offered by Allele), a fluorescent protein population can be created as in the aforementioned studies; but unlike bleaching or photoactivating, photoconversion keeps both populations (converted and unconverted, green or red in the case of mClavGR) present. This way all readings can be internally controlled to compensate for factors not directly related to protein metabolism per se, such as cell death, equipment variation, etc.

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Wednesday, February 16th, 2011 Fluorescent proteins No Comments

Many Thanks to Give

We would like to thank our customers for choosing Allele’s products and services. The economic conditions have been challenging for the last two years and are still difficult. Allele Biotech could not have possibly achieved what it has within this time period without the support of its customers.

We thank online readers who visit our Blogs, News and Technical Forum to make our webpage one of the highest ranked among biology reagent suppliers; surpassing Clontech, Stratagene, Promega, IDT, etc. (by online ranking service Alexa’s accounts).

We thank our collaborators (some converted from customers) and business partners (manufacturers, distributors, licensors) for working with Allele and making our plan of one new product per week a reality. Allele Biotech has a very innovative and able research team, but working with researchers outside of the company is always a major part of the R&D effort at Allele.

Allele Biotech is obliged to its employees for their creativity, organization, dedication, and professionalism. The culture that they have nurtured here is to be individually motivated as a basic research group, and to be disciplined and organized as a service provider at the same time.

Allele Biotech is a beneficiary of the US government’s policies on supporting biomedical research and innovation. We appreciate the support for our basic research in the forms of grants and contracts from the NIH and the IRS, and ultimately the American tax payers. It is our duty to create and produce better tools for improving human health.

Thank you once again and have a Happy Holiday!

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Wednesday, November 24th, 2010 Allele Mail Bag, Customer Feedback, Open Forum No Comments