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Abstract
Conventional protein affinity chromatography relies on highly porous resins that have large surface areas. These properties are ideal for fast flow separation of proteins from biological samples with maximum yields, but these properties can also lead to increased nonspecific protein binding. In certain applications where the purity of an isolated protein is more important than the yield, using a glass solid phase could be advantageous as glass is nonporous and hydrophilic and has a low surface area and low nonspecific protein binding. As a proof of principle, we used protein A-conjugated hollow glass microbubbles to isolate fluorescently labeled neurofilament heavy chain spiked into serum and compared them to protein A Sepharose and protein A magnetic beads (Dynabeads) using an anti-neurofilament protein antibody. As expected, a greater volume of glass bubbles was required to match the binding capacity of the magnetic beads and Sepharose resins. On the other hand, nonspecific protein binding to glass bubbles was greatly reduced compared to the other resins. Additionally, since the glass bubbles are buoyant and transparent, they are well suited for isolating cells from biological samples and staining themin situ.
Highlights
Glass is essentially an amorphous 3-dimensional mesh of silica oxides terminating at the surface as silicon hydroxides
Our results show that though glass bubbles have much lower binding capacity than Sepharose and Dynabeads, the glass bubbles had less nonspecific protein binding, as seen by the amount of total protein eluted and by SDS-PAGE protein profile
The glass microbubbles are very robust and remain intact in batch chromatography with end-over-end mixing, which allows for optimal contact of the bubbles with the analyte
Summary
Glass is essentially an amorphous 3-dimensional mesh of silica oxides terminating at the surface as silicon hydroxides. The surface exposed silica hydroxides readily coordinate with divalent cations such as Ca2+ and can promote surface activated plasma coagulation [1], glass is surprisingly resistant to protein adsorption. Glass bead chromatography has been used to purify vitronectin, a cell adhesion protein, from serum with a high degree of purity [2]. Vitronectin has an arginine-glycine-aspartic acid (RGD) integrin recognition motif and was initially identified as “serum spreading factor” due to its ability to bind tissue culture plates and mediate cell adhesion and spreading [3]. Development of porous polymer resins with superior flow characteristics and greater surface area have since replaced glass beads as the solid support of choice for most protein purification
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