Next-Generation RIPA Buffer for High Efficient Membrane Protein Extraction ULTRARIPA kit for Lipid Raft

(Data provided by Laboratory of Health Chemistry, Graduate School of Pharmaceutical Sciences, The University of Tokyo)

Composition of each buffer

• SDS buffer: 2％ SDS, 1％ Triton X-100, 50 mM HEPES・Na (pH 7.2), 150 mM NaCl
• ULTRARIPA kit A buffer: 1％ NP-40 Alternative, 0.1％ SDS, 50 mM Tris-HCl (pH8.0), 150 mM NaCl, 0.5％ Sodium Deoxycholate
• ULTRARIPA kit B buffer: undisclosed
• 1％ Triton X-100: 1％ Triton X-100, 50 mM HEPES・Na (pH 7.2), 150 mM NaCl

(Data provided by Department of PNS Research National Institute of Neuroscience, NCNP)

Sample: mouse primary cultured DRG neurons (DIV13, ～10⁶ cells/35 mm dish)
Target protein: Integrinβ1, Flotillin 1

Protocol:

Result:

Flotillin 1 is not changed by NGF stimulation. However, Integrin β1 is accumulated in RIPA-insoluble fraction by NGF stimulation.

(Data obtained in collaboration with Professor Akihiko Takashima and Assistant Professor Akio Sumioka, Factory of Science, Gakushuin University)

Sample: P2 membrane fraction^＊ derived from mouse brain tissue (hippocampus + cerebral cortex)
＊Protocol of extracting P2 membrane fraction

Composition of each buffer

• SDS: 2％ SDS, 50 mM Tris-HCl (pH 8.0), 150 mM NaCl
• 1％ Triton: 1％ TritonX-100, 50 mM Tris-HCl (pH 8.0), 150 mM NaCl
• ULTRARIPA kit A buffer (RIPA): 1％ NP-40 Alternative, 0.1％ SDS, 50 mM Tris-HCl (pH8.0), 150 mM NaCl, 0.5％ Sodium Deoxycholate
• ULTRARIPA kit B buffer: undisclosed

Protocol

Result

Sample: P2 membrane fraction derived from mouse brain tissue (hippocampus + cerebral cortex)

Protocol

Result

Sample: P2 membrane fraction derived from mouse brain tissue (hippocampus + cerebral cortex)
Target: NMDA glutamate receptor, NMDAR (GluN1/GluN2B)
　　　　AMPA glutamate receptor, AMPAR (GluA1/GluA2)

Protocol

Result

A-1 B-buffer has higher solubilization activity than RIPA (=A-buffer). This kit uses a two-step protocol to collect the RIPA-insoluble fraction and then dissolve it in B-buffer to concentrate and simply purify lipid raft proteins contained in the RIPA-insoluble fraction. When a sample is directly dissolved in B-buffer, the solubilization rate is higher than RIPA.

A-2 This product focuses on the fact that RIPA-insoluble fraction contains a lot of lipid raft proteins. By further solubilizing RIPA-insoluble fraction with B-buffer, it is mainly intended to solubilize lipid raft proteins in a non-denatured state. Therefore, even if proteins are not lipid rafts, if they are insoluble in RIPA buffer, they may be detected by this kit (see Q-3 for nuclear protein contamination).
This kit uses RIPA buffer with high solubilization capacity as A-buffer, however it is possible to replace RIPA buffer with another solubilizing buffer such as 1% TritonX-100.

A-3 This product uses a simple protocol to separate RIPA-soluble and RIPA-insoluble fractions by solubilizing A buffer and then centrifuging. Therefore, if unfractured cells/tissues or nuclei remain, RIPA-insoluble fraction may become contaminated. In particular, RIPA buffer has a weak nuclear solubilization efficiency, so there is a risk that a large amount of nuclei will remain in RIPA-insoluble fraction without physical fragmentation. On the other hand, B-buffer has a high nuclear solubilization efficiency, so if nuclei remain, genomic DNA will be released and the solution will become mushy. When solubilizing with A-buffer, sonication can destroy nuclei and fragment genomic DNA. The use of homogenization or sonication is strongly recommended because nuclear contamination can adversely affect subsequent applications.

A-4 Unfortunately, the composition of B-buffer and surfactant-type are not disclosed. However, our supplier has confirmed from various studies that B-buffer does not affect electrophoresis.

A-5 RIPA buffer can solubilize about 90~95% of total protein, including membrane proteins (depending on tissue and cell type). Therefore, in terms of total protein, the amount of protein contained in RIPA-insoluble fraction is very small, less than 10% of total protein. If RIPA-insoluble fraction cannot be observed visually, it is necessary to increase the number of cells or amount of tissue. If you want to increase the protein concentration in B-buffer soluble fraction, please reduce the volume of B-buffer added to RIPA-insoluble fraction. We recommend the following positive control experiment to consider the conditions.

Positive control experiment for considering starting-amount of sample
Things to prepare: 2% SDS buffer (50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 2% SDS)

Protocol:

A-6 We cannot guarantee that proteins detected as lipid raft markers by other methods will be obtained in A-buffer insoluble fraction. In particular, some lipid raft markers may vary depending on cell type, tissue, and the presence of external signals. Please refer to the following for specific examples.

Example 1: Insoluble in 1% TritonX-100, but detected in RIPA-buffer soluble fraction.
Improvement ideas: Because RIPA buffer (1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate) is more extractable than 1% TritonX-100, so target proteins may be solubilized in RIPA buffer. Using 1% TritonX-100 buffer instead of A-buffer(= RIPA buffer) may concentrate and solubilize the target proteins.

Example 2: Insoluble but not precipitated (in the case of insufficient centrifugation)
Improvement ideas: We recommend a centrifugal condition of 10,000×g or more, however it may not be sufficient depending on the target protein. In such cases, centrifuging at 20,000×g may precipitate the proteins. We recommend that you consider the centrifugal conditions.

A-7 Surfactant(s) in B-buffer can be removed by dialysis. The pore size of dialysis membrane should be around 5 kDa. However, some proteins may aggregate or denature when surfactant(s) is removed, so it may be necessary to add another surfactant(s) to dialysis buffer. It is recommended that a preliminary evaluation of dialysis be performed before the assay.