In the biological milieu of a cell, soluble crowding molecules and rigid confined environments strongly influence whether the protein is properly folded, intrinsically disordered proteins assemble into distinct phases, or a denatured or aggregated protein species is favored. Such crowding and confinement factors act to exclude solvent volume from the protein molecules, resulting in an increased local protein concentration and decreased protein entropy. A protein’s structure is inherently tied to its function. Examples of processes where crowding and confinement may strongly influence protein function include transmembrane protein dimerization, enzymatic activity, assembly of supramolecular structures (e.g., microtubules), nuclear condensates containing transcriptional machinery, protein aggregation in the contexts of disease and protein therapeutics. Historically, most protein structures have been determined from pure, dilute protein solutions or pure crystals. However, these are not the environments in which these proteins function. Thus, there has been an increased emphasis on analyzing protein structure and dynamics in more “in vivo-like” environments. Complex in vitro models using hydrogel scaffolds to study proteins may better mimic features of the in vivo environment. Therefore, analytical techniques need to be optimized for real-time analysis of proteins within hydrogel scaffolds.

在细胞的生物环境中，可溶性拥挤分子和刚性受限环境强烈影响蛋白质是否正确折叠、内在无序的蛋白质是否组装成不同的相，或者是否有利于变性或聚集的蛋白质种类。这种拥挤和限制因素的作用是从蛋白质分子中排除溶剂体积，导致局部蛋白质浓度增加和蛋白质熵降低。蛋白质的结构与它的功能有着内在的联系。拥挤和限制可能强烈影响蛋白质功能的过程的例子包括跨膜蛋白质二聚化、酶活性、超分子结构（例如微管）的组装、含有转录机制的核凝聚物、疾病和蛋白质治疗中的蛋白质聚集。从历史上看，大多数蛋白质结构都是从纯的、稀释的蛋白质溶液或纯晶体中确定的。然而，这些不是这些蛋白质发挥作用的环境。因此，越来越强调分析蛋白质结构和动力学的更多“体内类似”的环境。使用水凝胶支架研究蛋白质的复杂体外模型可以更好地模拟体内环境的特征。因此，需要优化分析技术以实时分析水凝胶支架内的蛋白质。