RIPA Lysis Buffer (Strong)

Introduction

RIPA Lysis Buffer is a traditional rapid cell tissue lysate used as the preferred lysate for protein extraction from tissues or cells in the Western Blot assay.

Components

Instructions

1.For Tissue Samples

a. Take the samples, wash the tissue thoroughly with pre-cooled PBS (0.01M, pH7.4) to remove the surface blood and internal debris.

b. Weigh and smash the tissue, add an appropriate ratio of RIPA Lysis Buffer (add 10 μL PMSF and 10 μL Na₃VO₄ to 1 mL RIPA Lysis) and homogenizely lyse the tissue.

It is recommended to homogenize according to the ratio of tissue weight: RIPA volume = 3:10. For example,add 1mL RIPA Lysis Buffer to 0.3 g tissue sample, the specific volume can be adjusted according to experimental requirements.

c. Shake and lyse on the ice for 30 min after homogenization. Blow the sample repeatedly with a Pipette gun for about 50 times (under ice water bath conditions) to Make sure the DNA strand is broken and reduce the viscosity of sample.
d. Centrifuge at 12,000 rpm for 10 min at 4°C.

e. Take the supernatant and measure the protein concentration.

2.For Cell Sample

a. Collect the cells, wash them thoroughly with pre-cooled PBS (0.01 M, pH7.4) to remove the medium off (it is generally recommended to wash 3 times).

b. Add an appropriate ratio of RIPA Lysate Buffer (add 10 μL PMSF and 10 μL Na₃VO₄ to1 mL RIPA Lysis) and lyse on the ice for 30 min.

It is recommended to add 0.1 mL of RIPA Lysis Buffer to each well of a 6-well plates (the protein content in different cells may vary, and the volume of the RIPA Lysis Buffer added can be appropriately adjusted).

c. Blow the sample repeatedly with a Pipette gun for about 50 times (under ice water bath conditions) to Make sure the DNA strand is broken and reduce the viscosity of sample. 

d. Centrifuge at 12,000 rpm for 10 min at 4°C.

e. Take the supernatant and measure the protein concentration.

RIPA Lysis Buffer components

50 mM Tris (pH7.4), 150 mM NaCl, 1% Triton X-100, 1% C₂₄H₃₉O₄Na, 1 mM EDTA, 0.1% SDS, 10 mM NaF, 1 mM Na₃VO₄, 1 mM PMSF.

Storage

Store at -20°C for 12 months.

Cautions 

1. For the protein extraction with RIPA Lysis Buffer, the whole process must be on the ice or at 4°C.

2. It is recommended to add 10 μL PMSF and 10 μL Na₃VO₄ to RAPI Lysis before use. If RIPA Lysis is crystalline state,dissolve at room temperature or in a warm water bath.

3. A cloud of transparent gels may appear in the RIPA Lysis product,  which is a normal phenomenon because the lysis product is a compound containing genomic DNA.Blow the sample repeatedly with a Pipette gun for about 50 times, It can effectively reduce the sample viscosity,Take the supernatant after centrifugation for subsequent testing.

4.The protein sample lysed by RIPA cannot be measured by the Bradford method because of the high concentration of detergent in the lysis. It is recommended to measure the protein concentration by the BCA method.

5. For your safety and health, please wear the lab coat and disposable gloves before the experiments.

Citations