Next-Generation RIPA Buffer for High Efficient Membrane Protein Extraction ULTRARIPA kit for Lipid Raft
Date：November 14 2017Web Page No：80549
RIPA buffer is one of the most useful buffers for protein extraction. RIPA buffer maintains most native structures of proteins and the extracted proteins can be applied to various applications. However, RIPA buffer is not sufficient to extract membrane proteins and membrane-associated proteins concentrated in lipid raft.
Lipid raft is a highly specialized microdomain on the lipid bilayer which contains specialized lipids, cholesterol and functional proteins. These lipid rafts are also called “Detergent Resistant Membrane (DRM)”, as lipid raft-enriched proteins are usually insoluble by mild detergent buffers such as 1% Triton X-100 and RIPA buffer. Consequently, it was difficult to analyze functions of lipid raft-enriched proteins extracted with RIPA buffer.
BioDynamics Laboratory's newly developed product:the ULTRARIPA kit, can efficiently and rapidly extract membrane proteins or membrane-associated proteins enriched in lipid rafts, with little effect on protein structures and functions.
ULTRARIPA kit includes a totally new buffer not containing protein denaturing detergents, but can extract the DRM which was difficult to extract by conventional RIPA buffer. ULTRARIPA kit helps to analyze various biological assays of the proteins in lipid raft.
What is “ Lipid Raft ”?
Lipid raft is a highly specialized microdomain on the lipid bilayer which contains special lipids, cholesterol and functional proteins.
Example of Lipid Raft :Caveolae, Synapse (Neuron), Immunological synapse
Problems with conventional extraction method
Lipid Rafts are also called as “Detergent Resistant Membrane (DRM)”. Lipid Rafts are usually insoluble by mild detergent buffers such as 1% Triton X-100 and RIPA buffer. SDS can solubilize Lipid Rafts, but the extracted proteins are not suitable for functional assay due to SDS’s strong denaturation ability.
Consequently, it was difficult to analyze functions of lipid raft-associated proteins extracted by these buffers.
ULTRARIPA kit can help you!
- Two extraction buffers solubilize Lipid Rafts and concentrate
- Less protein denature = Mild extraction
- Easy handling : just adding buffers and centrifugation
Features of ULTRARIPA kit
- Extract membrane / membrane associated proteins enriched in lipid rafts, with little effect on protein structures and functions.
- Easy and Simple Procedure : Only a centrifuge is required
- Only two components: A buffer and B buffer
- A buffer (RIPA Buffer) : 100 mL
- B buffer : 10 mL
Outline of procedureExtract Lipid Raft proteins in 30 minutes!
- Solubilize samples (tissues / cells) by A Buffer (RIPA)
- Centrifuge (>10,000 x g, 5 minutes) and collect A buffer insoluble fraction
- Add B buffer to A buffer insoluble fraction
- Centrifuge (>10,000 x g, 5 minutes) and collect soluble fraction
- Use it for further assays!
Overview of advantages of ULTRARIPA kit
|SDS Buffer||RIPA Buffer||ULTRARIPA Kit|
|Cytoplasm proteins||Can extract but proteins are denatured||Can extract non-denatured proteins||Can extract non-denatured proteins|
|Membrane proteins (non-Lipid Raft)||Can extract but proteins are denatured||Can extract non-denatured proteins||Can extract non-denatured proteins|
|Membrane proteins (Lipid Raft)||Can extract but proteins are denatured||Cannot extract||Can extract non-denatured proteins|
|Immunoprecipitation of Lipid Raft proteins||Not suitable||Not suitable||Suitable|
|Enzyme activity assay of Lipid Raft proteins||Not suitable||Not suitable||Suitable|
Extraction and functional analysis of the Lipid Raft proteins from mouse brain
Sample:Mouse whole brain
Procedure:Solubilize RIPA-insoluble fraction by each buffer
Extracting proteins from RIPA-insoluble fraction
>70% extracted proteins from RIPA-insoluble fraction was observed compared to 2% SDS extraction buffer.
Extraction of Lipid Raft marker (Caveolin-1 and GM-1)
Caveolin-1 and GM-1 were efficiently extracted.
Antigen-Antibody reaction or Antibody-Protein A/G reaction are not affected
⇒ Compatible with immunoprecipitation
Phosphatase activity assay
Enzyme activity is maintained
⇒ Compatible with enzyme activity assay for RIPA insoluble fraction (≒Lipid Raft protein)
Extraction efficiency by direct addition of B-buffer to sample
Sample:P2 membrane fraction of mouse brain tissue (hippocampus + cerebral cortex)
Procedure:1. Solubilize P2 membrane fraction by each buffer
2. Solubilize each insoluble fraction by adding 2%SDS
B-buffer can extract most part of synaptic proteins compared to 1% Triton X100 or RIPA buffer (A-buffer).
Data : Acquired under the support of Professor Akihiko Takashima and Research associate Dr. Akio Sumioka at Department of Life Science, Gakushuin University.
Analysis of synaptic-protein complex
Immunoprecipitation by using B-buffer soluble fraction
- Physiological NMDAR or AMPAR complex is specifically detected by Immunoprecipitation
- Best for analysis of protein complex in RIPA insoluble fraction (≒Lipid Raft protein)
NGF stimulation dependent migration of Integrin to lipid raft
Sample:Mouse primary cultured DRG neurons
Procedure:Following to ULTRARIPA kit protocol
Flotillin 1 is not changed by NGF stimulation. However, Integrin β1 is accumulated in RIPA-insoluble fraction by NGF stimulation
Data provided by Department of PNS Research National Institute of Neuroscience, NCNP
Not only detecting the change of RIPA insoluble fraction dependent on external stimulus, but also...
- Detecting the change of protein complex dependent on external stimulus
- Detecting the change of enzyme activity in RIPA insoluble fraction
[Date : January 26 2021 23:29]
|Detail||Product Name||Product Code||Supplier||Size||Price|
ULTRARIPA kit for Lipid Raft
||F015||BDLBioDynamics Laboratory Inc||1 kit||$120|
[Date : January 26 2021 23:29]
ULTRARIPA kit for Lipid Raft
- Product Code: F015
- Supplier: BDL
- Size: 1kit
- Price: $120
ULTRARIPA kit, can efficiently and rapidly extract membrane proteins or membrane-associated proteins enriched in lipid rafts with native structure and function.
- ※Prices are for USA / Canada customers. Prices do not include shipping and handling charges, VAT, import tariffs and service charge etc.
- ※Please note that Product Information or Price may change without notice.