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Next Generation RIPA

A Next-Generation RIPA Buffer with High Efficiency of Membrane Protein Extraction

UltraRIPA Buffer

UltraRIPA Product Appearance

Highlights

  • High Efficiency of Membrane Protein Extraction: 2% SDS > UltraRIPA > RIPA > 1% Triton X-100
    More than 70% of protein is solubilized from RIPA-insoluble fraction.

  • Low Protein Denaturation Effect: 2% SDS >> UltraRIPA ≒ RIPA ≒ 1% Triton X-100
    Extracted protein by UltraRIPA buffer can be used for enzymatic activity assay and immunoprecipitation.

Product Information

UltraRIPA buffer, an improved RIPA buffer, provides significantly high protein extraction efficiency with low denaturation effect. Proteins extracted by UltraRIPA buffer largely maintain their native structure and biological activity and can be applied to various applications. Some specific membrane proteins such as lipid raft proteins are hardly solubilized by conventional non-denaturing cell lysis buffers such as RIPA buffer and 1% Triton X-100. UltraRIPA buffer is suitable for functional analysis of the Triton X-100- / RIPA-insoluble proteins.

Application

Comparison of Protein Solubilization Capacity

 

 

 

Protein Extraction Efficiency

Experimental Procedure
A mouse brain was lysed with RIPA buffer and homogenized by a dounce tissue grinder and sonicator. After centrifugation of the lysate, 2% SDS buffer, RIPA buffer, or UltraRIPA buffer was added to the RIPA-insoluble pellet and resuspended it well. Each supernatant after centrifugation was applied to silver stain and BCA protein assay.

Experimental Result
More than 70% of protein was extracted from RIPA-insoluble fraction using UltraRIPA buffer.

Comparison of Enzyme Activity in Extracted Samples

 

 

 

Enzymatic Activity

Experimental Procedure
CHO cells were directly lysed in 1% Triton X-100 containing buffer, RIPA buffer, UltraRIPA buffer, or 2% SDS buffer. After centrifugation, Lactate Dehydrogenase (LDH) activity in each supernatant was measured.

Experimental Result
LDH activity in supernatant extracted with UltraRIPA buffer was equivalent to those in Triton X-100 containing buffer and RIPA buffer.

Application 1:Comparison of Protein Extraction Effiency from COS-1 cell

Experimental Procedure
COS-1 cells were lysed in SDS buffer, 1% Triton X-100, RIPA buffer, or UltraRIPA buffer. The lysates were separated into soluble and insoluble fractions by centrifugation (14,000 rpm, 5 min, 4°C). The insoluble fraction was resuspended and denatured in an equal volume of SDS-PAGE sample buffer. The solubilized amount of Flotilin1, one of lipid raft markers, was evaluated by SDS-PAGE/Western blot.

Composition of Each Buffer
・SDS buffer (2% SDS, 1% Triton X-100, 50 mM HEPES・Na (pH 7.2), 150 mM NaCl)
・RIPA buffer (1% NP-40 Alternative, 0.1% SDS, 50 mM Tris-HCl (pH8.0), 150 mM NaCl, 0.5% Sodium Deoxycholate)
・UltraRIPA buffer
・1% Triton X-100 buffer (1% Triton X-100, 50 mM HEPES・Na (pH 7.2), 150 mM NaCl)

 

 

 

Comparison of extraction effiency from COS-1 cell

Experimental Result
In 1% Triton X-100 and RIPA buffer, most of Flotilin1 remained in insoluble fraction, whereas almost all of Flotilin1 was solubilized in UltraRIPA buffer.

Application 2:Comparison of Protein Extraction Effiency from Mouse P2 Membrane Fraction

Experimental Procedure
P2 membrane fraction was prepared from mouse brain tissue (hippocampus + cerebral cortex). 100 μl of each buffer was added to P2 fraction and sonicated on ice. Each solution was centrifuged at high speed (x100,000 g) and collected each soluble fraction. The insoluble fraction was solubilized by adding 100 μl of 2% SDS buffer.

Composition of Each Buffer
・SDS buffer (2% SDS, 50 mM Tris-HCl (pH 8.0), 150 mM NaCl)
・RIPA buffer (1% NP-40 Alternative, 0.1% SDS, 50 mM Tris-HCl (pH8.0), 150 mM NaCl, 0.5% Sodium Deoxycholate)
・UltraRIPA buffer
・1% Triton X-100 buffer (1% Triton X-100, 50 mM Tris-HCl (pH 8.0), 150 mM NaCl)

Comparison of extraction effiency from mouse P2 membrane fraction

Experimental Result
UltraRIPA Buffer showed higher solubilization efficiency than 1% Triton X-100 and RIPA buffer for all tested neuronal synapse-associated proteins.

 

 

 

Product Name
Product Code
Size
Manual
SDS
UltraRIPA Buffer
100 mL
Product Name
UltraRIPA Buffer
Product Code
Size
100 mL
Manual


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