RIPA Lysis Buffer High Efficiency for Tissue and Cell

Product Introduction

RIPA Lysis Buffer is a traditional rapid lysis solution for cells and tissues. It comes in various formulations, primarily categorized as strong, medium, and weak based on their lysis intensity. The strong RIPA Lysis Buffer is suitable for lysing tissues and cells to obtain protein samples for conventional applications such as PAGE and Western blot, where strict protein activity preservation is not required. The main components of this product include 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA-2Na, 1% Triton X-100, 1% sodium deoxycholate, and 0.1% SDS. It is applicable for animal or plant tissues and cell samples, and can also be used for fungal or bacterial samples.

Product Specification

This product utilizes a substance with reducing properties comparable to dithiothreitol (DTT) or β-mercaptoethanol (which has a strong odor), while being odorless and exhibiting improved stability in aqueous solutions. Its performance demonstrates no significant difference from conventional SDS-PAGE protein loading buffers, thereby making protein loading procedures safer and healthier.

Storage

Transported with ice packs (wet ice); stored at -20°C, valid for 12 months.

Operation Step

Prepare protease inhibitors separately. Protease inhibitors must be added to the RIPA Lysis Buffer (Strong) immediately before use. G2006, G2007, G2008, or similar products are recommended to prevent protein degradation. In the following procedures, "RIPA Lysis Buffer (Strong)" refers to the buffer already supplemented with protease inhibitors.

For Tissue Samples:
1. Rinse the tissue piece with pre-cooled PBS to remove blood contaminants, and mince it into fine fragments in a homogenizer.

2. Add 10 volumes (relative to tissue mass) of RIPA Lysis Buffer (Strong) and homogenize at low temperatures ( high-speed tissue homogenizer YB-HL is recommended). Note: The amount of RIPA Lysis Buffer (Strong) used can generally follow a ratio of approximately 500 µL per 50 mg of tissue. If the tissue has low protein content, the amount of lysis buffer can be reduced to increase the protein concentration in the crude extract.

3. Transfer the homogenate to a 1.5 mL centrifuge tube and vortex. Incubate on ice for 30 minutes, pipetting up and down repeatedly every 10 minutes to ensure complete lysis of tissue cells.

4. Centrifuge at 12,000×g for 5 minutes. Collect the supernatant, which is the total protein solution.

For Adherent Cell Samples:

1. Wash the cells with PBS 2-3 times, thoroughly aspirating the residual liquid after the final wash.

2. Add RIPA Lysis Buffer (Strong) to the cell culture plate or flask at a ratio of 250 µL per well of a 6-well plate. Gently rock the plate or flask to ensure the lysis buffer fully contacts the cells for 3-5 minutes.

3. Scrape the cells off using a cell scraper and collect them into a centrifuge tube.

4. Lyse the cells on ice for 30 minutes.

5. Centrifuge at 12,000 × g for 5 minutes. Collect the supernatant, which is the total protein solution.

For Suspension Cell Samples:

1. Collect the cells by centrifugation.

2. Mix the cell pellet with RIPA Lysis Buffer (Strong) at a ratio corresponding to 250 µL per well of a 6-well plate, and vortex.

3. Incubate on ice for 30 minutes, pipetting up and down several times every 10 minutes to ensure complete cell lysis.

4. Centrifuge at 12,000×g for 5 minutes. Collect the supernatant, which is the total protein solution.

For Bacterial or Fungal Samples:

1. Take 1 mL of microbial suspension, centrifuge to remove the supernatant, wash once with PBS, and thoroughly remove the liquid. Vortex to disperse the pellet as much as possible.

2. Add 100-200 µL of RIPA Lysis Buffer (Strong) and gently vortex to thoroughly mix the microbial cells with the lysis buffer.

3. Incubate on ice for 10 minutes, pipetting up and down several times every 2 minutes to ensure complete microbial lysis.

4. Centrifuge at 12,000×g for 5 minutes. Collect the supernatant, which is the total protein solution.

Notice

1. During tissue or cell lysis, the lysate may become viscous. Repeatedly pipette the mixture or vortex it until it becomes liquid. If it remains overly viscous, add an appropriate amount of additional lysis buffer.

2. This reagent does not contain protease inhibitors. It is necessary to prepare protease inhibitors separately and add them immediately before use. We recommend using our company's related protease inhibitors.

3. Please wear a lab coat and disposable gloves during operation.