RIPA Lysis Buffer is a traditional rapid lysis buffer for cells and tissues. There are many formulations of RIPA (Radio Immunoprecipitation Assay) lysis buffer, which are mainly divided into three types based on their lysis efficiency: weak,
medium, and weak. Protein samples obtained from tissues and cells lysed with RIPA Lysis Buffer (weak) can be used for Western Blot, IP, Co-IP, and ELISA. The main components of this product include 50 mM Tris-HCl (pH 7.4),
150 mM NaCl, 1 mM EDTA-2Na, 0.25% Sodium deoxycholate, and 1% NP-40. This product is suitable for animal or plant tissue and cell samples, and can also be used for fungal or bacterial samples.
Protease inhibitors need to be prepared by the user. Protease inhibitors must be added to RIPA Lysis Buffer (weak) immediately before use. Add different protease inhibitors according to the sample type to prevent protein degradation.
1. Wash the tissue pieces with pre-cooled PBS to remove blood contamination, cut them into small pieces, and place them in a homogenizer.
2. Add 10 volumes of RIPA Lysis Buffer (weak) based on the tissue volume and homogenize at low temperature.
Note: The amount of RIPA Lysis Buffer (weak) to use can be added at a ratio of approximately 1 mL of lysis buffer 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 solution.
3. Transfer the homogenate to a 1.5 mL centrifuge tube and vortex. Incubate on ice for 30 min. During incubation, pipette up and down every 10 min to ensure complete lysis of tissue cells.
4. Centrifuge at 12,000 g for 5 min. Collect the supernatant, which is the total protein solution.
1. Wash the cells 2-3 times with PBS. Thoroughly aspirate the residual liquid after the last wash.
2. Add RIPA Lysis Buffer (weak) to the cell culture plate or flask at a ratio of 250 μL of lysis buffer per well of a 6-well plate. Gently rock the plate or flask to ensure full contact between the lysis buffer and cells for 3-5 min.
3. Scrape the cells off using a cell scraper and collect them into a centrifuge tube.
4. Lyse on ice for 30 min.
5. Centrifuge at 12,000 g for 5 min. Collect the supernatant, which is the total protein solution.
1. Collect the cells by centrifugation.
2. Mix the cell pellet with RIPA Lysis Buffer (weak) at a ratio of 250 μL of lysis buffer per well of a 6-well plate. Vortex.
3. Incubate on ice for 30 min. During incubation, pipette up and down several times every 10 min to ensure complete cell lysis.
4. Centrifuge at 12,000 g for 5 min. Collect the supernatant, which is the total protein solution.
1. Take 1 mL of the cell suspension, centrifuge and remove the supernatant. Wash once with PBS and thoroughly remove the liquid. Vortex to disperse the cells as much as possible.
2. Add 100-200 μL of RIPA Lysis Buffer (weak). Gently vortex to mix the cells thoroughly with the lysis buffer.
3. Incubate on ice for 10 min. During incubation, pipette up and down several times every 2 min to ensure complete cell lysis.
4. Centrifuge at 12,000 g for 5 min. Collect the supernatant, which is the total protein solution.
1. The lysate may become viscous during tissue or cell lysis. Pipette up and down repeatedly or vortex until it becomes liquid. If it remains viscous, add an appropriate amount of additional lysis buffer.
2. This reagent does not contain protease inhibitors. Protease inhibitors must be prepared by the user and added immediately before use.
3. Please wear a lab coat and disposable gloves during operation.
4. This product is for research use only and is not intended for clinical diagnosis or treatment.
