Here, self-assembling peptide hydrogels (SAPHs) are of particular interest. The term hydrogel describes a well-hydrated, porous scaffold which can be fabricated from an array of natural and/or synthetic polymers. Recently, hydrogels have become increasingly popular as biomaterials used to encapsulate cells for a range of applications, from 3-D cell culture to in vivo cell delivery ( 10, 11). Determining how these biomaterials might interfere with different analytical techniques is important in selecting and modifying current protocols to obtain accurate and reliable results. As a result, a variety of new biomaterials have appeared in the literature that allow the 3-D encapsulation of cells ( 9). In the past two decades, there has been a move from 2-D to 3-D cell culture methods, with a view to recapitulate the native 3-D cellular niche in vitro. Sample-specific optimization is an intrinsic part of method development. Typical buffer components include detergents (anionic, cationic, and non-ionic), salts, reducing agents, pH-buffering compounds, and protease and phosphatase inhibitors ( 2). The choice of buffer depends on sample type ( 5–7), protein of interest ( 8), and downstream application. In general, extracting proteins from cell samples relies on the ability to lyse cells in a buffer that promotes protein solubilization. As with any technique, the accuracy of the results depends heavily on the sample preparation and, in this case, specifically on the recovery and solubilization of cellular proteins for downstream analysis ( 4). The term “western blot” is used to describe the entire process from protein extraction, solubilization, and size separation through to membrane transfer and subsequent antibody detection ( 3).
Over the last 40 years ( 1), western blot analysis has become a wide-spread technique used for a range of purposes, including identification and quantification of proteins, determination of protein–protein interactions, and detection of post-translational proteins modifications, to name a few ( 2). Moreover, we show that the presence of self-assembling peptides (SAPs) does not interfere with the standard immunoblotting technique, offering the potential for use in more full-scale proteomic studies. We demonstrate that both the choice of buffer and multiple cycles of sonication are vital in obtaining complete solubilization, thereby enabling the detection of proteins otherwise lost to SAP aggregation. Here, we describe an approach for western blot analysis of cells that have been encapsulated in self-assembling peptide hydrogels (SAPHs), which highlights the need for complete solubilization of the hydrogel construct. In particular, isolation of cellular components for downstream applications is often hindered by the presence of biomaterials, presenting a major obstacle in understanding how cell–matrix interactions influence cell behavior. Continuous optimization of in vitro analytical techniques is ever more important, especially given the development of new materials for tissue engineering studies.
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