Background of Lysosome Purification by Free Flow Electrophoresis
The biochemical and physical properties of lysosomes often overlap, thus requiring fractionation techniques capable of detecting small differences in density, charge, or composition for efficient separation. Free Flow Electrophoresis (FFE) exploits differences in the overall charge of bio-particles separating cells, organelles, macromolecules, ions, etc. according to their distinct electrophoretic mobility and isoelectric point values. In recent years this technique has been applied to subcellular membrane binding Separation of organelles. The unique feature of the negatively charged surface of the lysosome that deflects it towards the anode makes it the first organelle successfully purified by FFE. In addition, the surprising electrophoretic heterogeneous mobility of lysosomes was confirmed, suggesting that FFE has great potential in separating lysosomes.
Fig. 1. Schematic overview of sample fractionation by Free Flow Electrophoresis (FFE) and downstream analysis by mass spectrometry. (Guo Q, et al., 2021)
Our Free Flow Electrophoresis Lysosome Purification Services
FFE is based on separation of surface charge density of organelles. Based on the applied electric field, lysosomes migrate in a specific pattern and are purified. Over the years, CD BioSciences has developed several different lysosome purification techniques. Here, our engineers developed a FFE method to enrich lysosomes from cells and tissues. We have used FFE not only to purify lysosomes from non-polarized cells such as human diploid skin fibroblasts and small hamster kidney cells, but also to purify endosomes (which are organs of lysosomal precursor cells). We optimized the previous protocol by three aspects:
- Trypsinization of the preisolates obtained by differential centrifugation.
- Percoll density gradient centrifugation of the combined endonucleosomal-lysosomal pool.
- Incorporation of endocytic tracers to monitor endosome purification and identify specific target populations.
Here, our engineers developed a rapid and efficient procedure that allows for the analysis and preparation of isolated nuclear endosomes from a variety of tissue culture cells.
(1) We used endo-horseradish peroxidase, FITC-coupled dextrose, and methionine-labeled Semliki Forest virus as markers of endosomes.
(2) Total postnuclear peroxisomes, crude granule pellets or partially purified Golgi fractions were subjected to FFE.
(3) Endosomes and lysosomes migrate together as a single anode-deflected peak, separated from most other organelles.
(4) Lysis of endosomes and lysosomes by Percoll density gradient centrifugation.
Why Choose Us
- The purification process takes only 5-6 hours.
- FFE is an efficient method for analysis and preparation.
- FFE can be cooled, run in a vertical position, and ensure stable flow conditions.
- The FFE technique relies on different surface charges, rather than the buoyant density of organelles, and best supports secondary separation of lysosomes based on centrifugation.
- Adequate numbers of endosomes can be generated for biochemical, immunological and functional analysis.
- Lysosomal structure is well preserved and verified by labeled enzymes and electron micrographs.
- Endosomes prepared by this method are enriched up to 70-fold.
CD BioSciences can meet any reasonable needs of our clients, taking time and budget into consideration for you. Our aim is to be customer-centric and to provide the highest quality services to customers. Our customer service representatives are enthusiastic and trustworthy 24 hours a day, 7 days a week. If you are interested in our services, please feel free to contact us for more information or a detailed discussion.
References
- Guo Q, Liu L, Yim W C, et al. (2021) Membrane profiling by Free Flow Electrophoresis and SWATH-MS to characterize subcellular compartment proteomes in Mesembryanthemum crystallinum[J]. International journal of molecular sciences. 22(9): 5020.
- Marsh M. (1989) Endosome and lysosome purification by free-flow electrophoresis[M]//Methods in cell biology. Academic Press, 31: 319-334.