ULTRARIPA kit for Lipid Raft. 1. Basic information

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1 ULTRARIPA kit for Lipid Raft 1. Basic information

2 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

3 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 Mild buffer Denaturing buffer However RIPA buffer cannot fully dissolve proteins from cells and tissues.

4 Problem of RIPA buffer For example : Lysis of mouse brain tissue by RIPA buffer RIPA buffer After Lysis After centrifugation After wash Ms brain + RIPA buffer w/ Homogenizer w/ Sonication RIPA-sol RIPA-insol RIPA-insol <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. Only supernatants are available for following assays. = So far, RIPA insoluble fractions are regarded as the cell debris!

5 2% SDS RIPA-sol RIPA-insol +2%SDS 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-insoluble mainly Lipid raft proteins RIPA-soluble centrifugation ~95% RIPA-soluble mainly Cytosolic proteins Non-raft membrane proteins ~5% These proteins Cannot be extracted by RIPA buffer Can be extracted by SDS buffer with strong denaturing condition RIPA-insoluble Although RIPA-insoluble fraction contains functionally important proteins, But we cannot access these functions RIPA-soluble RIPA-insol RIPA insoluble fraction-enriched proteins

6 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

7 Overview of lipid rafts 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. Sectional view of the lipid raft Example of lipid rafts: Biologically significant structures

8 How many proteins are identified in Lipid raft? RaftProt database of lipid raft research 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. RIPA-sol Solubilized insoluble fraction by SDS buffer And MS analysis Identified ~ 8,000 proteins = There are a lot of functionally unknown proteins Black box of proteins

9 Functional proteins in lipid rafts Various functional proteins were discovered from lipid rafts!! Protein family Total In RaftProt Ratio Protein Kinase % Protein phosphatase % Phosphatase % GPCR % Transporter % Ion Channel 280 RIPA-sol 56 20% Integrin % Lipase/esterase % * All members, not only membrane protein, were searched % of functional proteins family were identified from lipid raft

10 Current subject of mild extraction buffer Lipid Raft contains - About 8,000 proteins - A lot of functionally significant proteins/enzymes Treasure-trove of proteins SDS buffer RIPA buffer RIPA insoluble Fraction = Lipid raft To access to functions of RIPA-insoluble proteins (=Lipid raft proteins), We need more attractive mild extraction buffers!

11 ULTRARIPA kit for Lipid Raft 2. Application note

12 New Solution : ULTRARIPA buffer ULTRARIPA buffer - Is next generation mild extraction buffer - Contains non-denaturing detergent - Has higher extraction activity than RIPA buffer Overview of UltraRIPA buffer Protein extraction Buffer Cytosolic proteins Nonlipid raft Membrane proteins Lipid raft Protein Structure Protein Function Application SDS buffer SDS-PAGE RIPA buffer Enzymatic assay Binding assay (IP etc.) SDS-PAGE ULTRARIPA kit etc.

13 Overview of ULTRARIPA kit for Lipid Raft ULTRARIPA kit for Lipid raft The kit is optimized to analyze Lipid raft proteins. Component of ULTRARIPA kit A buffer (RIPA buffer) B buffer (ULTRARIPA buffer) 100 ml 10 ml 1. Isolation of RIPA-insoluble fraction by A-buffer 2. Effective extraction of lipid raft proteins by B-buffer ULTRARIPA-insol tightly insoluble proteins RIPA-sol RIPA-insol >70% <30% ~95% purification ~5% ULTRARIPA-sol For various applications because of less-denaturing condition - Analysis of protein complex Immunoprecipitation - Measurement of enzymes each enzymatic assays

14 Protocol of ULTRARIPA kit Easy and quick extraction of Lipid raft proteins 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 ULTRARIPA kit A buffer (RIPA buffer) B buffer (ULTRARIPA buffer)

15 ULTRARIPA kit can efficiently extract lipid raft proteins Validation 1: How much proteins can ULTRARIPA kit extract from RIPA-insoluble fractions? <Experiment with a mouse brain> - Silver staining - BCA assay (protein quantification) - Western Blotting against lipid raft markers Positive control 2% SDS buffer Negative control ULTRARIPA kit A-buffer ULTRARIPA kit B-buffer ULTRARIPA kit A buffer (RIPA buffer) B buffer (ULTRARIPA buffer) Over 70% of proteins are extracted from RIPA-insoluble fraction by ULTRARIPA buffer

16 ULTRARIPA can be applicable to immunoprecipitation Validation 2: Can ULTRARIPA kit be compatible with binding assays such as immunoprecipitation assay? <Example with a mouse brain> ULTRARIPA kit B-buffer ULTRARIPA -sol ULTRARIPA-insol UltraRIPA kit A buffer (RIPA buffer) B buffer (ULTRARIPA buffer) PSD-95: multi-interactive protein in neurons ULTRARIPA can be useful for immunoprecipitation There are possibilities of detection of unknown and novel protein complexes

17 ULTRARIPA has little effects on enzymatic activities Validation 3: Can ULTRARIPA buffer be compatible with enzymatic activity measurements? <Experiment with cultured cells> Effect of UltraRIPA buffers on lactose dehydrogenase (LDH) activity * One of the common assay to measure influences on enzymatic activity Positive control 1% TritonX100 buffer ULTRARIPA kit A-buffer ULTRARIPA kit B-buffer UltraRIPA kit A buffer (RIPA buffer) B buffer (UltraRIPA buffer) Negative control 2% SDS buffer ULTRARIPA kit ULTRARIPA kit A and B buffers have no effect on LDH activity ULTRARIPA kit are able to apply for enzymatic activity assays

18 Detection of enzymatic activities of lipid raft enzymes Validation 4: Can ULTRARIPA kit detect any enzymatic activities of Lipid Raft Proteins? <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) Positive control 2% SDS buffer Protein conc. (mg/ml) Phosphatase activity Negative control ULTRARIPA kit A-buffer ULTRARIPA kit B-buffer 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

19 Direct extraction by ULTRARIPA buffer How about 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.

20 Stimuli-dependent translocation of raft protein Sample : mouse primary cultured DRG neurons (DIV13) *This experimental data provided by Department of PNS Research, National Institute of Neuroscience (NCNP), Japan

21 Extraction of synaptic proteins (1) 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

22 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

23 Analysis of synaptic protein complexes Sample : mouse brain-derived membrane fraction (P2 fraction) P2 fraction Add 200 ml of B-buffer Collect sup Immunoprecipitation (IP) *This experimental data was obtained under cooperate research with Prof. Akihiko Takashima and Dr. Akio Sumioka, Gakushuin University