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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.

Lipid raft and Detergent Resistant Membrane (DRM)
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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

MEMO What is  Lipid Raft


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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.

Problems with conventional extraction method


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ULTRARIPA kit can help you!



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

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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

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Kit components

  • A buffer (RIPA Buffer) : 100 mL
  • B buffer : 10 mL
ULTRARIPA kit for Lipid Raft component


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Outline of procedure

Extract Lipid Raft proteins in 30 minutes!
  1. Solubilize samples (tissues / cells) by A Buffer (RIPA)
  2. Centrifuge (>10,000 x g, 5 minutes) and collect A buffer insoluble fraction
  3. Add B buffer to A buffer insoluble fraction
  4. Centrifuge (>10,000 x g, 5 minutes) and collect soluble fraction
  5. Use it for further assays!

Procedure of ULTRARIPA kit


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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


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Extraction and functional analysis of the Lipid Raft proteins from mouse brain

Sample:Mouse whole brain
Procedure:Solubilize RIPA-insoluble fraction by each buffer

ultraripa kit protocol

Result

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.

ultraripa kit result

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Immunoprecipitation

ultraripa kit immunoprecipitation

Antigen-Antibody reaction or Antibody-Protein A/G reaction are not affected
⇒ Compatible with immunoprecipitation

Phosphatase activity assay

ultraripa phosphatase activity assay

Enzyme activity is maintained
⇒ Compatible with enzyme activity assay for RIPA insoluble fraction (≒Lipid Raft protein)


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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

ultraripa kit protocol

Result
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.

ultraripa kit immunoprecipitation

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Analysis of synaptic-protein complex
Immunoprecipitation by using B-buffer soluble fraction

ultraripa kit analysis of synaptic protein complex
  • Physiological NMDAR or AMPAR complex is specifically detected by Immunoprecipitation
  • Best for analysis of protein complex in RIPA insoluble fraction (≒Lipid Raft protein)

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NGF stimulation dependent migration of Integrin to lipid raft

Sample:Mouse primary cultured DRG neurons
Procedure:Following to ULTRARIPA kit protocol

Result

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

ultraripa kit NGF

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

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Product Information

[Date : September 16 2021 23:25]

Detail Product Name Product Code Supplier Size Price
ULTRARIPA kit for Lipid Raft
DatasheetThis may not be the latest data sheet.
F015 BDLBioDynamics Laboratory Inc 1 kit $140

Description ULTRARIPA kit, can efficiently and rapidly extract membrane proteins or membrane-associated proteins enriched in lipid rafts with native structure and function.
Storage 4°C CAS
Link

[Date : September 16 2021 23:25]

ULTRARIPA kit for Lipid Raft


  • Product Code: F015
  • Supplier: BDL
  • Size: 1kit
  • Price: $140

Description ULTRARIPA kit, can efficiently and rapidly extract membrane proteins or membrane-associated proteins enriched in lipid rafts with native structure and function.
Storage 4°C CAS
Link

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CONTACT

export@funakoshi.co.jp

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