For Research Use Only. Not for Clinical Use.

The evaluation of half-life is critical for the success of drug development. To most therapeutics, such as protein, peptides, small molecule drugs, and so on, longer half-lives are desired. Therefore, the half-life extension is often involved in the development of such drugs. The determination of whether the half-lives of the drugs have been successfully extended and how long the half-lives of drugs are essential in this context. Creative Biolabs has a comprehensive technology platform offering various drug half-life in vitro detection services.

Drug Half-Life In Vitro Detection Platform at Creative Biolabs

• Half-life Assay by Pulse-chase

The pulse-chase analysis encompasses a way to assay the rate of degradation of specific protein fragments and has been used for several years to monitor protein degradation in several mammalian cultured cells including numerous neuronal cell lines.

• Half-life Assay by Cycloheximide Chase

In CHX-chase analysis, the administration of a global protein translation inhibitor (such as cycloheximide, CHX) to block protein translation is performed (CHX-pulse phase). This is followed by examining the reduction in the levels of a target protein chase via sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting.

Fig. 1 Pulse-chase and cycloheximide (CHX)-chase assays. (Eldeeb, 2019)

• Half-life Assay by Photoactivatable Fluorescent Proteins (PAFP)

This method provides a unique opportunity to observe real-time target protein degradation at the single-cell level without the addition of protein synthesis inhibitors, to follow conditionally dependent changes in degradation rate, and to correlate this with cell cycle stage, morphology, and other characteristics of the particular cells.

• Half-life Assay by Proteomic Approaches

Proteomics-based assays for studying proteins half-lives can be divided into two types: (a) Dynamic stable isotope labeling in cell culture (SILAC). The use of different isotopic versions of an amino allows the temporal tracking of protein abundance. (b) Pulse-chase-coupled with SILAC. A pulse-chase assay coupled with SILAC can be performed to study the degradation kinetics of a large number of proteins.

• Half-life Assay by The Ubiquitin-reference Technique

The ubiquitin reference technique (URT) approach relies on a fusion in which the Ubiquitin (Ub) is positioned in the middle between an upstream metabolically stable reference protein and a downstream test protein. Upon expression in cells, endogenous ubiquitin hydrolases mediate the co-translational cleavage of a URT fusion after the last residue of Ub to generate a test protein fragment with a desired N-terminal amino acid residue and a reference protein that encompasses the C-terminal Ub moiety.

• Half-life Assay by The Promoter-reference Technique

One of the biochemical techniques that have been developed recently to monitor protein degradation in the eukaryotic system is the promoter-reference technique (PRT). This method has been exploited to identify the Pro-N-end rule pathway, a novel proteolytic pathway in eukaryotic cells that targets protein fragments harboring N-terminal proline.

• Half-life Assay by Fluorescent

Commonly, there are used two methods for tracking protein degradation using fluorescent proteins (FPs). (a) Protein fluorescent switchers. FPs can exhibit features of photoactivation, photoswitching, or photo converting, where a distinct population of labeled proteins is produced using a specific wavelength and intensity of light. (b) Protein fluorescent timers. A Fluorescent timer can report on the average age of a pool of protein fragments.

• Half-life Assay by Bleach-chase

This elegant non-invasive assay depends on the fact that a brief pulse of light renders a fluorescent tag non-fluorescent, in an irreversible manner. This tag reversal is only partial and creates two subpopulations (fluorescent and non-fluorescent) within a protein of interest in the cell. In this case, the protein removal or disappearance rate is tracked as the difference between bleached and unbleached protein fluorescence. As such, the protein half-life is determined.

• Half-life Assay by Biolayer Interferometry

Through the biolayer interferometry method, researchers determined a high-throughput in vitro model capable of predicting the human half-life of naturally occurring, fully human IgG mAbs. This model performs similar to in vivo models with transgenic FcRn mice and was used to predict features that may influence mAb half-life, including complementarity-determining regions (CDRs) and Fc region glycosylation.

With a comprehensive technology platform and extensive experience accumulated from years of practice, Creative Biolabs is confident in offering high-quality drug half-life in vitro detection services to global customers. If you are interested in our services or looking for assistance in drug half-life in vitro detection, please feel free to contact us for professional solutions from a team of scientists.

Reference

1. Eldeeb, M. A.; et al. A molecular toolbox for studying protein degradation in mammalian cells. J Neurochem. 2019, 151(4): 520-533.

Related Services:

• Half-life Assay by Pulse-chase
• Half-life Assay by Cycloheximide Chase
• Half-life Assay by Photoactivatable Fluorescent Proteins (PAFP)
• Half-life Assay by Proteomic Approaches
• Half-life Assay by The Ubiquitin-reference Technique
• Half-life Assay by The Promoter-reference Technique
• Half-life Assay by Fluorescent
• Half-life Assay by Bleach-chase
• Half-life Assay by Biolayer Interferometry