Description

Lipid rafts are small membrane domains containing high level of cholesterol and sphingolipids. Lipid rafts are found in plasma membrane (PM) and internal organellar membranes such as mitochondria-associated membrane (MAMs) and endoplasmic reticulum. They have been implicated in numerous cellular processes including but not limited to signal transduction, membrane trafficking and protein sorting. Lipid-modified proteins and some transmembrane proteins are concentrated in the rafts while other proteins are excluded. Lipid rafts are also found to be associated with Na+/K+ ATPase on plasma membrane. Lipid rafts are traditionally isolated by sucrose-gradient or OptiPrep gradient using ultracentrifugation that requires large amount of starting material. The protocol is tedious and time consuming. This kit was developed using our proprietary spin-column-based technologies, offering a rapid and easy way to isolate lipid rafts. Total membrane fraction (include PM and organelle membranes) is first isolated and treated with a non-ionic detergent containing buffer, followed by isolation of detergent resistant fraction by flotation centrifugation using just a table top microcentrifuge. Highly enriched total lipid rafts can be obtained from cultured cells/tissues in less than 90 min without using density gradient and ultracentrifugation.



*For isolation of plasma membrane-drived lipid rafts, please refer to MinuteTM Plasma Membrane-Drived Lipid Raft Isolation Kit under Cat #: LR-042





Image 1: Lipid rafts floating/ Isolated Lipid Rafts

Image 2: Evaluation of Cross-Contamination of Isolated Lipid Rafts

Lipid rafts were isolated from mouse (TR, left) and human (right) cortex that were probed with H3 and GAPDH. No presence of either in the lipid raft fraction. Human crude nuclear extract has some GAPDH, likely from RBCs. Data: Courtesy of Dr. Max Thorwald, University of Southern California.


Image 3: Detection of Lipid Raft Marker in Isolated Lipid Rafts

Lipid raft fractions obtained from isolation kit for mouse (TR, left) and human cortex (right) were probed with Flotillin-1. Lipid raft fractions are positive for Flotillin-1. Data: Courtesy of Dr. Max Thorwald, University of Southern California.


Image 4: Nitrocellulose blot stained with Ponceau

Kit Includes

References

1. Kim, S., Choi, Y., Kwon, C., & Yun, H. S. (2018). Endoplasmic reticulum stress‐induced accumulation of VAMP721/722 requires CALRETICULIN 1 and CALRETICULIN 2 in Arabidopsis. Journal of integrative plant biology.

2. Yang, Z., Yang, J., Wang, Y., Wang, F., Mao, W., He, Q., ... & Mao, C. (2020). PROTEIN PHOSPOHATASE 95 Regulates Phosphate Homeostasis by Affecting Phosphate Transporter Trafficking in Rice. The Plant Cel

3. Yu, F., Cao, X., Liu, G., Wang, Q., Xia, R., Zhang, X., & Xie, Q. (2020). ESCRT-I Component VPS23A Is Targeted by E3 Ubiquitin Ligase XBAT35 for Proteasome-Mediated Degradation in Modulating ABA Signaling. Molecular Plant, 13(11), 1556-1569.

4. Kato, T., Morita, R., Ootsuka, S., Wakabayashi, Y., Aoki, N., & Horibata, A. Evaluation of alleles at OsAGPS2, OsAGPL2, and OsSUT1 related to grain filling in rice in a common genetic background. Crop Science.

5. Hou, L. Y., Lehmann, M., & Geigenberger, P. (2021). Thioredoxins o1 and h2 Show Different Subcellular Localizations and Redox-Active Functions, but Cooperatively Affect NADPH Redox Poise and Photosynthetic Performance in Fluctuating Light.

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