NIST mAb is a widely characterized monoclonal antibody designed to be used as a standard in the development of new protein characterization technologies. In this application literature, we use SUPR-DSF to obtain differential scanning fluorescence (DSE) instructions for NIST mAb, and compare the results with those obtained by scanning calorimetry (DSO). SUPR-DSE has resolved all two domains of NISTAb: CH2 (690C. CH3 (830())) and the FAB domain with high solubility (94oC) [1]. In addition, the measured Tm values are consistent with those reported in the literature. These results demonstrate that the application of SUPR-DSF can easily obtain high-quality protein stability information.

Conclusion:

Protein Stable's SUPR-DSF can detect the peak shift of intrinsic fluorescence in proteins and quickly and accurately measure the melting temperature. The results indicate that SUPR-DSF can obtain accurate melting temperatures, including ultra-high melting temperatures such as Fab domains (94 ° C). This also confirms that SUPR-DSF based on endogenous fluorescence method can obtain results consistent with DSC. The advantage of using a 384 well plate for testing is that it improves flux, increases convenience, and reduces sample consumption.

Method:

NIST mAb with a concentration of 1mg/ml was prepared in L-histidine buffer at pH 6 and added to 384 well LPCR microplates with a dosage of 104L per well. After packaging, the microplates were centrifuged for 60 seconds and sealed with a transparent film. Set the continuous up mixing program in SUPR-DSF to measure the fluorescence spectra from 150C to 105oC at a heating rate of 10C per minute. The data obtained from the fluorescence spectra will be calculated and processed using the BCM method, and the spectral shift will be quantified. The data from BCM will be processed as a function of temperature and converted into a first-order derivative for further analysis. Calculate the melting temperature based on the peak value of the derivative. All methods and results of the samples can be obtained.