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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)