Journal: Journal of Cell Science
Article Title: Rapid affinity purification of intracellular organelles using a twin strep tag
Figure Lengend Snippet: Determination of factors critical to lysosome purification. (A) Effect of bead sizes using beads from Creative Diagnostics. The same amount of PNS was incubated with streptavidin beads of different diameters as indicated for 5 min. The volume of the beads for each size was adjusted to have the same binding capacity. Lysosome abundance in the purified products was determined by western blotting for LAMP2, showing that 1 µm diameter beads are the most efficient. (B) Comparison of the effect of bead sizes on lysosomal purification efficiency using beads from Thermo Fisher Scientific. Western blot analyses of LAMP2 from PNS and products of purification of Lyso-2Strep using 25 µm and 1 µm streptavidin beads. (C) Purity of the lysosome preparation. Western blot analyses of PNS and products after purification with Lyso-2Strep using 1 µm streptavidin beads. Intracellular organelle markers used were: LAMP1 and LAMP2 (lysosomes, Lyso), SDHA and TOM20 (mitochondria, Mito), SLC27A2 and catalase (peroxisomes, Pex), PDI and SERCA (endoplasmic reticulum, ER), Golgin-97 and GOLM1 (Golgi), ATP1A1 and PMCA2 (plasma membrane, PM), S6K and ERK1/2 (cytosol, Cyt). The purified lysosomes are free of contamination from other organelles. (D) Intactness of the lysosome preparation. Fluorescence images of lysosomes isolated from HeLa cells expressing Lyso-2Strep (green), eluted from streptavidin beads with 20 mM biotin and then labeled with LysoTracker (red). Scale bar: 20 μm. (E) Effect of incubation time and bead abundance. The same amount of PNS (100 µl) was incubated with 30 µl or 60 µl of 1 µm diameter streptavidin beads (Thermo Fisher Scientific) for different time periods as indicated. The relative recovery efficiency of lysosomes was determined by comparing the density of LAMP2 to that of PNS on the same western blots.
Article Snippet: Antibodies for S6K (cat. #9202, 1:1000), SDHA (cat. #5839, 1:1000), catalase (cat. #12980, 1:1000), PDI (cat. #3501, 1:1000), Golgin-97 (cat. #13192, 1:1000) and ERK1/2 (cat. #4696, 1:1000) were from Cell Signaling Technology (Danvers, MA); antibody for SERCA (cat. #SC-271669, 1:1000) was from Santa Cruz Biotechnology (Dallas, TX); antibody for LAMP1 (cat. #L1418, 1:2500) was from Sigma-Aldrich (St Louis, MO); antibodies for PMCA2 (cat. #19678-1-AP, 1:1000), ATP1A1 (cat. #14418-1-AP, 1:1000), GOLM1 (15126-1-AP, 1:1000), SLC27A2 (14048-1-AP, 1:1000) and TOM20 (cat. #11802-1-AP, 1:10,000) were from ProteinTech (Rosemont, IL); and antibody for LAMP2 (cat. #9840-01, 1:2500) was from the Developmental Studies Hybridoma Bank (Iowa City, IA).
Techniques: Purification, Incubation, Binding Assay, Western Blot, Fluorescence, Isolation, Expressing, Labeling