Background of Flux Measurement of Purified Lysosomes
Ion channels convert various signals into transmembrane ion fluxes. Each channel has different effector activities based on different functional characteristics, as well as control of expression and localization. Currently, cytoplasmic membrane ion channels and intracellular subcellular ion channels have become common targets for toxins and therapeutic agents. Developments in sensor technology and instrumentation have enabled the measurement of new ion channels in a higher throughput manner. Concentrated isotope uptake was used to measure ion fluxes of ion channels in membrane vesicles. Researchers have successfully incubated vesicle suspensions with NaCl isotopes to measure the amount of Na captured within the vesicles. Therefore, we attempted to apply this method to the measurement of lysosomal ion channels.
Fig. 1. Fluorescence-based membrane potential assay format for detecting potassium channel inhibitors. (McManus OB, et al., 2012)
Flux Measurement Services of Purified Lysosomes
Our biologists incorporated lysosomal ion channels into lipid vesicles (liposomes) and characterized the functional characteristics of the lysosomal ion channel population by measuring radioisotope uptake in these proteoliposomes. Here at CD BioSciences, we focus on providing our global customers with an efficient flux measurement service for purified lysosomes to estimate fluxes in large numbers of lysosomes.
We develop customized processes for fluorescence measurement of lysosomal CLC-7 ion channels using focused Cl- uptake binding proton fluxes.
(1) Lysosomes were isolated from rat liver loaded with high concentrations of unlabeled chloride by differential sedimentation and then diluted into a buffer containing Cl-.
(2) Rapid uptake of Cl- was eliminated by external addition of valinomycin.
(3) Alteration of internal anions and additional experiments in the presence of a pH gradient to establish the apparent permeability sequence.
(4) Measurements were performed using a Cl- gradient and the internal pH was monitored using the fluorescent dye 2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF).
(5) The equilibrium potential of the H+ flux monitored by BCECF was measured at a set theoretical voltage.
Why Choose Us
- Allows measurement of endolysosomal ion channels in the native environment.
- This method allows estimation of fluxes in a large number of lysosomes under different external conditions and keeps the protein in its natural membrane.
- The ion transport mechanism across the lysosomal membrane can be identified.
- Requires radiometric measurements and does not allow direct and precise control of membrane potential.
- Flux measurements require 2-5 hours depending on the reaction rate and the number of time points require.
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.
Reference
- McManus OB, et al. (2012) Ion Channel Screening. Assay Guidance Manual [Internet].