Perform a quality control experiment to determine which fractions are enriched with lysosomes. Activity and protein analysis can be performed on neat samples taken from Percoll fractions. For example, analyze each fraction by Western blot, probing for the lysosome marker LAMP1 (Figure 1B). Analysis of the cation independent mannose-6-phosphate receptor (CI-M6PR) is useful as this receptor is present on endosomes but not lysosomes (Cheng
et al., 2018), evident in fractions 13-18. Additionally, activity of lysosomal enzymes (such as β-hexosaminidase, β-glucocerebrosidase, β-galactosidase, and acid phosphatase) can be used to detect lysosomal fractions (Figure 1C) (Leaback and Walker, 1961; Kolodny and Mumford, 1976). If cell lysis was too vigorous and lysosomes were burst open, this will be revealed as a ‘flat’ β-hexosaminidase profile across the fractions as opposed to a profile that peaks in the denser fractions. Cell components other than endosomes and lysosomes will be present in this gradient as well. For example, plasma membrane will be enriched in the lightest fractions (Perret
et al., 1979). Endoplasmic reticulum will also appear in lighter fractions, and the Golgi apparatus will appear in intermediate fractions. Mitochondria may appear in heavier fractions and need to be considered as potential contaminants when purifying lysosomal fractions (Suhy
et al., 2000).
Figure 1. Density-gradients yield subcellular fractions that are enriched with lysosomes. A. Schematic of experimental workflow. Briefly, cells are gently lysed by popping in a syringe and the soluble fraction layered on top of a Percoll/sucrose cushion. Ultra-centrifugation for 1 h at 30,000
x g at 4 °C will distribute organelles along the gradient in a density-dependent manner. Top-to-bottom fractionation is used to separate organelles based on their sedimentation profile. Less dense organelles (such as early endosomes) sediment toward the top of the gradient, whereas denser organelles (such as late endosomes and lysosomes) sediment toward to bottom of the gradient. B. Western blot analysis of density-gradient fractions (#1-19; out of 19) from HeLa cells. Blots were probed for the late endosome/lysosome marker LAMP1. Note that LAMP1 is enriched in high-density fractions #13-18. C. Activity analysis of lysosomal enzymes across density-gradient fractions (#1-16; out of 16). Note that for all enzymes there is increased activity in high-density fractions #13-15. Data presented is the mean of three independent experiments.
Figure 2. Preparation of a continuous Percoll gradient. A. Homogenized cell sample is placed into an ultracentrifuge tube. B. 18% Percoll is placed underneath the homogenized sample. C. Before ultracentrifugation, cell lysate should be seen as a discreet layer (black arrow) on top of the Percoll suspension. D. After centrifugation, the gradient appears relatively featureless, although high-density Percoll may be observed as a cloudy region or ‘button’ at the bottom of the tube.