A high-throughput differential scanning fluorimetry method for rapid detection of thermal stability and iron saturation in lactoferrin.

Thermal stability and iron saturation of lactoferrin (LF) are of great significance not only for the evaluation of the biological activities of LF but also for the optimization of the isolation and drying process parameters. Differential scanning calorimetry (DSC) is a well-established and efficient method for thermal stability and iron saturation detection in LF. However, multiple DSC measurements are typically performed sequentially, thus time-consuming and low throughput. Herein, we introduced the differential scanning fluorimetry (DSF) approach to overcome such limitations. The DSF can monitor LF thermal unfolding with a commonly available real-time PCR instrument and a fluorescent dye (SYPRO orange or Glomelt), and the measured melting temperature of LF is consistent with that determined by DSC. On the basis of that, a new quantification method was established for determination of iron saturation levels using the linear correlation of the degree of ion saturation of LF with DSF measurements. Such DSF method is simple, inexpensive, rapid (<15 min), and high throughput (>96 samples per experiment), and provides a valuable alternative tool for thermal stability detection of LF and other whey proteins.

Aptamer-mediated rolling circle amplification for label-free and sensitive detection of histone acetyltransferase activity.

We develop for the first time an aptamer-mediated rolling circle amplification approach for label-free and sensitive detection of histone-modifying enzyme (HME) activity. This method can achieve femtomolar sensitivity for histone acetyltransferase Tip60 assay, which is the most sensitive HME assay reported so far. It can be further applied for inhibitor screening, enzyme kinetic analysis, and endogenous Tip60 measurement in cancer cells.