Background of Lysosome Purification by Immunoprecipitation
Advances in analytical techniques have made the need for isolated organelle analysis even more important. Immunoprecipitation (IP) is a method for small-scale affinity purification of antigens using specific antibodies immobilized on solid-phase supports such as magnetic beads or agarose resin. Scientists have successfully developed affinity purification and immunoprecipitation strategies for lysosomal isolation. A variety of antibodies directed against the A or B structural domains of the V1 subunit of the vesicular H+-ATPase (V-ATPase) pump allow efficient isolation of pure lysosomes suitable for histological analysis.
Fig. 1. Widely used techniques for lysosome isolation include differential centrifugation, Lyso-IP, and SPIONs. (Chen C, et al., 2022)
Our Immunoprecipitation Lysosome Purification Services
Methods by V-ATPase pumps are limited by the need for long incubations with V-ATPase antibodies, the use of sucrose extraction buffers that interfere with mass spectrometry analysis, and the use of large numbers of multiple antibodies. For many years, CD BioSciences has developed several different lysosome purification techniques. Here, our engineers developed Lyso-IP to purify lysosomes.
The principle of Lyso-IP is to use immobilized antibodies to bind antigens present on the surface of lysosomes with high specificity and strength. Our IP-based lysosome purification service focuses on the immunoprecipitation of intact lysosomes by adding them to epitopes in the cytoplasmic fraction of the following lysosomal proteins. We will develop the best solution for you based on the characteristics of these two strategies.
- Overexpression of tagged lysosomal membrane proteins
We fuse the triple human influenza virus hemagglutinin (3×HA) epitope tag with lysosomal transmembrane protein 192 (TMEM192), TMEM192-3×HA, allowing for rapid isolation of lysosomal anti-HA magnetic beads. The characteristics are as follows:
- Relative speed, allowing purification of lysosomes in less than ten minutes.
- No previous processing is required.
- Requires appropriate surface exposure for antigen, or overexpression of marker proteins, combined with appropriate beads and antibodies.
- Allows recovery of lysosomal proteins only, not intact organelles.
- Isolated lysosomes retain lysosomal protease activity and contain many lysosomal luminal proteins.
- Bistreptococcal tag and streptavidin variants
We purified lysosomes from cells expressing LAMP1 mGFP 2xStrep fusion protein using a high affinity bistreptococcal tag and strep affinity variant. The characteristics are as follows:
- Allows for more rapid purification.
- Less starting material is required to produce the same amount of purified lysosomes.
- Purification of different organelles from the same sample.
Why Choose Us
- Lyso-IP provides significantly better proteome coverage of lysosomes compared to the gradient density centrifugation method.
- Lyso-IP significantly increases the purification of specific lysosomal protein markers compared to organelle-rich precipitation separations.
- Lyso-IP increases the known lysosomal proteins in the lysosomal fraction compared to whole cell lysates.
- Lyso-IP isolation of lysosomes is one of the best methods for label-free quantitative data.
- Lysosomes isolated by Lyso-IP have been successfully used for proteomics, lipidomics and metabolomics analyses.
- Lyso-IP provides new insights into the pathogenic mechanisms of lysosomal diseases.
- Lyso-IP can be easily applied to different animal models of disease.
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
- Chen C, Sidransky E, Chen Y. (2022) Lyso-IP: Uncovering Pathogenic Mechanisms of Lysosomal Dysfunction[J]. Biomolecules. 12(5): 616.