ULTRARIPA kit for Lipid Raft

1. Basic information

Background: Cell lysis buffers

SDS-containing buffer

Advantages

- Strong extraction activity

⇒ Fully extraction of cells

Disadvantages

- Denaturing protein structure

- Not compatible with protein functional analysis

Mild cell extraction buffers

ex.RIPA buffer, 1% Triton X-100

Advantages

- Keep protein structures

- Keep protein functions

- Compatible with protein functional analysis

⇒ Enzymatic activity assay kit etc.

Disadvantages

- Not fully extraction of tightly membrane-attached proteins

- It is hardly to analyze functions of protein in the insoluble

fraction

What is RIPA buffer?

RIPA (Radioisotope ImmunoPrecipitation Assay) buffer

- Composition

50 mM Tris-HCl (pH 8.0), 150 mM NaCl,

+ 1% NP-40, 0.1% SDS, 0.5% Sodium Deoxycholate

- Applications

For… enzymatic assay, immunoprecipitation assay, etc.

- One of the most popular buffer for long time

- One of the strongest buffer among mild buffers

1% NP-40

1% Triton X-100 < RIPA buffer << SDS buffer

1% Deoxycholate

However…

RIPA buffer cannot fully dissolve proteins from cells and tissues.

Mild buffer Denaturing buffer

Problem of RIPA buffer

After Lysis After centrifugation After wash

RIPA-sol

RIPA-insol RIPA-insol

RIPA buffer

＜An example of RIPA buffer protocol from company X＞1. Add cold RIPA Buffer to the cells. Keep on ice for 5 minutes, swirling the plate occasionally for uniform

spreading.

2. Gather the lysate to one side using a cell scraper, collect the lysate and transfer to a microcentrifuge tube.

Centrifuge samples at ~14,000xg for 15 minutes to pellet the cell debris.

3. Transfer supernatant to a new tube for further analysis.

For example : Lysis of mouse brain tissue by RIPA buffer

Only supernatants are available for following assays.

= So far, RIPA insoluble fractions are regarded as the cell debris!

Ms brain+ RIPA buffer

w/ Homogenizer

w/ Sonication

What proteins are enriched in RIPA insoluble fractions?

RIPA buffer enable to extract ~95% of total protein.

However, some proteins are completely remained in the RIPA insoluble fractions.

When cells/tissues are lysed by RIPA buffer

RIPA-solublemainly…

・ Cytosolic proteins

・ Non-raft membrane proteins

～95%

～5%

RIPA-insoluble

RIPA-soluble

centrifugation

RIPA insoluble

fraction-enriched

proteins

RIPA-insoluble

mainly Lipid raft proteins

These proteins ･･･Cannot be extracted by RIPA buffer

Can be extracted by SDS buffer with strong

denaturing condition

Although RIPA-insoluble fraction contains

functionally important proteins,

But… we cannot access these functions

RIPA-soluble

RIPA-insol2

% S

DS

RIP

A-s

ol

RIP

A-insol

+2%

SD

S

RIPA-insoluble fraction may be Lipid rafts

A lot of studies indicate RIPA-insoluble fractions are “Lipid rafts”.

What is lipid rafts?

In 1987, Dr. Kai Simons proposed that plasma membranes are not homogenous.

It is heterogeneous structure which has microdomains. ⇒ Mosaic lipid raft model

Example of lipid rafts:

Biologically significant structures

Sectional view of the lipid raft

Lipid rafts contains…

- specialized lipids such as sphingolipids and cholesterol

- lipidated proteins such as GPI-anchor proteins, palmitoylated proteins

Based on these structures, various types of functional proteins are accumulated in lipid rafts.

Overview of lipid rafts

How many proteins are identified in Lipid raft?

RIPA-sol

RaftProt – database of lipid raft research

http://lipid-raft-database.di.uq.edu.au/index.html

provided by Queensland University

RaftProt is a database which has proteomics data from 81 papers.

Now,

"7959 proteins”are resistered as lipid raft enriched proteins.

Identified

~ 8,000 proteins

Solubilized insoluble fraction by SDS buffer

And MS analysisThere are a lot of functionally

unknown proteins

“Black box of proteins”

=

Functional proteins in lipid rafts

RIPA-sol

Various functional proteins were discovered from lipid rafts!!

Protein family Total In RaftProt Ratio

Protein Kinase 516 112 22%

Protein phosphatase 148 34 23%

Phosphatase 90 27 30%

GPCR 412 17 4%

Transporter 509 183 36%

Ion Channel 280 56 20%

Integrin 26 10 38%

Lipase/esterase 134 31 23%

20-30% of functional proteins family were identified from lipid raft

* All members, not only membrane protein, were searched.

Current subject of mild extraction buffer

RIPA insoluble

Fraction

= Lipid raftSDS buffer RIPA buffer

Lipid Raft contains

- About 8,000 proteins

- A lot of functionally

significant proteins/enzymes

Treasure-trove

of proteins

To access to functions of RIPA-insoluble proteins (=Lipid raft proteins),

We need… more attractive mild extraction buffers!

ULTRARIPA kit for Lipid Raft

2. Application note

New “Solution” : ULTRARIPA buffer

Buffer

Protein extraction

Protein

Structure

Protein

FunctionApplication

Cytosolic

proteins

Membrane

proteins

Non-

lipid raftLipid raft

SDS buffer ○ ○ ○ × × SDS-PAGE

RIPA buffer ○ ○ × ○ ○ Enzymatic assay

Binding assay (IP etc.)

SDS-PAGE

etc.ULTRARIPA kit ○ ○ ○ ○ ○

ULTRARIPA buffer…- Is next generation mild extraction buffer

- Contains non-denaturing detergent

- Has higher extraction activity than RIPA buffer

Overview of UltraRIPA buffer

Overview of ULTRARIPA kit for Lipid Raft

Component of ULTRARIPA kit

A buffer (RIPA buffer) 100 ml

B buffer (ULTRARIPA buffer) 10 ml

ULTRARIPA kit for Lipid raft

The kit is optimized to analyze Lipid raft proteins.

RIPA-solRIPA-insol

purification

ULTRARIPA-sol

>70%

<30%

ULTRARIPA-insol

tightly insoluble proteins

1. Isolation of RIPA-insoluble fraction

by A-buffer2. Effective extraction of lipid raft proteins

by B-buffer

For various applications because of less-denaturing condition

- Analysis of protein complex ⇒Immunoprecipitation

- Measurement of enzymes ⇒each enzymatic assays

~95% ~5%

Protocol of ULTRARIPA kit

1. Cell or tissues are lysed by A-buffer

2. Centrifuge at 14,000 rpm for 5 min

3. Remove supernatant and the pellet is washed by A-buffer

4. Centrifuge at 14,000 rpm for 5 min and remove supernatant

5. Add B-buffer to the RIPA-insoluble pellet and suspended

6. Centrifuge at 14,000 rpm for 5 min

7. Collect supernatant

To following assays…

(10 min)

(5 min)

(1 min)

(5 min)

(1 min)

(5 min)

Total ~30 min

Easy and quick extraction of Lipid raft proteins

ULTRARIPA kit

A buffer (RIPA buffer)

B buffer (ULTRARIPA buffer)

ULTRARIPA kit can efficiently extract lipid raft proteins

Over 70% of proteins are extracted from RIPA-insoluble fraction by ULTRARIPA buffer

＜Experiment with a mouse brain＞- Silver staining

- BCA assay (protein quantification)

- Western Blotting against lipid raft markers

Validation 1：How much proteins can ULTRARIPA kit extract from RIPA-insoluble fractions?

ULTRARIPA kit

A buffer (RIPA buffer)

B buffer (ULTRARIPA buffer)

Negative control

ULTRARIPA kit

A-buffer

ULTRARIPA kit

B-buffer

Positive control

2% SDS buffer

ULTRARIPA can be applicable to immunoprecipitation

＜Example with a mouse brain＞

ULTRARIPA can be useful for

immunoprecipitation

There are possibilities of detection of

unknown and novel protein complexes

Validation 2：Can ULTRARIPA kit be compatible with binding assays such as

immunoprecipitation assay?

UltraRIPA kit

A buffer (RIPA buffer)

B buffer (ULTRARIPA buffer)

PSD-95:

multi-interactive protein in neurons

ULTRARIPA kit

B-buffer

ULTRARIPA-insol

ULTRARIPA

-sol

ULTRARIPA has little effects on enzymatic activities

＜Experiment with cultured cells＞Effect of UltraRIPA buffers on lactose dehydrogenase (LDH) activity

* One of the common assay to measure influences on enzymatic activity

ULTRARIPA kit A and B buffers

have no effect on LDH activity

ULTRARIPA kit are able to apply for

enzymatic activity assays

Validation 3：Can ULTRARIPA buffer be compatible with enzymatic activity measurements?

UltraRIPA kit

A buffer (RIPA buffer)

B buffer (UltraRIPA buffer)

ULTRARIPA kit

A-buffer

ULTRARIPA kit

B-buffer

ULTRARIPA kit

Negative control

2% SDS buffer

Positive control

1% TritonX100 buffer

Detection of enzymatic activities of lipid raft enzymes

＜Experiment with a mouse brain＞Measurement of phosphatase activity in lipid raft

※protein phosphatase family

about 140 member (23% family members are registered in RaftProt)

Validation 4：Can ULTRARIPA kit detect any enzymatic activities of “Lipid Raft Proteins”?

Protein conc. (mg/ml)Phosphatase activity

UltraRIPA kit

A buffer (RIPA buffer)

B buffer (ULTRARIPA buffer)

ULTRARIPA can detect protein

phosphatase activity from lipid rafts

ULTRARIPA kit has potent possibility of detection

of unannotated enzymatic activity from lipid rafts

ULTRARIPA kit

Negative control

ULTRARIPA kit

A-buffer

ULTRARIPA kit

B-buffer

Positive control

2% SDS buffer

Direct extraction by ULTRARIPA buffer

Flotilin-1, one of the most popular lipid raft marker protein, was efficiently solubilized by

ULTRARIPA buffer compared with conventional 1% TritonX-100 and RIPA buffer.

<Procedure of direct extraction>

RIPA

Sol<Ppt

ULTRARIPA

Sol>>Ppt

1% TritonX100

Sol<Ppt

*This experimental data was provided by Tokyo University, Prof. Arai laboratory.

How about direct extraction by ULTRARIPA buffer?

Stimuli-dependent translocation of raft protein

*This experimental data provided by Department of PNS Research,

National Institute of Neuroscience (NCNP), Japan

Sample : mouse primary cultured DRG neurons (DIV13)

Extraction of synaptic proteins (1)

*This experimental data was obtained under cooperate research with Prof. Akihiko Takashima and Dr. Akio Sumioka,

Gakushuin University

Sample : mouse brain-derived membrane fraction (P2 fraction)

Extraction of synaptic proteins (2)

Sample : mouse brain-derived membrane fraction (P2 fraction)

*This experimental data was obtained under cooperate research with Prof. Akihiko Takashima and Dr. Akio Sumioka,

Gakushuin University

Analysis of synaptic protein complexes

Sample : mouse brain-derived membrane fraction (P2 fraction)

*This experimental data was obtained under cooperate research with Prof. Akihiko Takashima and Dr. Akio Sumioka,

Gakushuin University

P2 fraction

Add 200 mL of B-buffer

Collect sup

Immunoprecipitation (IP)