Abstract
Cell-free extracts from unfertilized Xenopus laevis eggs offer the opportunity for a variety of biochemical and biophysical assays for analyzing essential cell cycle events such as metaphase spindle assembly. However, the extracts often exhibit substantial variation in quality and have low storage stability, factors that hamper their experimental utility. Here we report a simple two-step method for preparing frozen egg extracts that retain spindle assembly activity levels similar to those of freshly prepared extracts. Extract degradation associated with the freeze-thaw process can be substantially reduced by using centrifugal filter-based dehydration and slow sample cooling. Large amounts of frozen extract stocks from single-batch preparations allowed us to collect extensive data in micromanipulation experiments, which are often low-throughput, and thus enabled the clarification of correlations between metaphase spindle size and stiffness. Our method provides an assay platform with minimized biological variability and improves the accessibility of egg extracts for research.