RIPA lysis buffer (medium)

Product Description
RIPA (Radio Immunoprecipitation Assay) lysis buffer is a classic, rapid lysis solution for animal tissues and cells. By combining ionic detergents with non-ionic detergents, it efficiently disrupts cellular structures, releasing proteins while effectively lysing cell membranes, cytoplasmic subfractions, and nuclear components.
Li Ji Bio RIPA Lysis Buffer (Medium) PMSF-free formulation ; Suitable for research applications including PAGE, Western blotting, and immunoprecipitation (IP).
Order Information

Transportation and Storage
Store at room temperature. Store at 4°C; shelf life: 12 months.
Formula List
25 mM Tris•HCl, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, pH 7.6
Instructions for Use
Adherent cells
1. Add an appropriate amount of lysis buffer, along with a protease inhibitor mixture (100×, EDTA-free) (Catalog No.: AP02L084) or PMSF (final concentration of…).
1 mM, but PMSF is insufficient to inhibit all protease activities).
2. Remove the cell culture medium and wash twice with pre-cooled PBS. Add 150–200 µL of lysis buffer per well of a 6-well plate, distributing it evenly.
Add the lysis buffer; if the cell density is too high, increase the volume of lysis buffer to 250–300 µL. [Note]: Insufficient lysis buffer may result in incomplete cell lysis.
Sufficiently, it affects the quality of protein extraction.
3. Blow with the gun several times, then place on ice for 10 minutes, blowing with the gun every 2 minutes during this period.
4. Transfer the cell lysate into a 1.5 mL EP tube.
5. Centrifuge at 12,000 g and 4°C for 5 minutes.
6. Collect the supernatant.
Suspension cells
1. Add an appropriate amount of lysis buffer, along with a protease inhibitor mixture (100×, EDTA-free) (Catalog No.: AP02L084) or PMSF (final concentration of…).
1 mM, but PMSF is insufficient to inhibit all protease activities).
2. Collect 0.5–2 × 10^7 cells in a 1.5 mL centrifuge tube, wash twice with 1 mL of pre-cooled PBS, centrifuge at 1,000 × g for 3 min, and discard the supernatant.
Repeat three times.
3. Add lysis buffer uniformly at a ratio of 150–200 µL per well in a 6-well plate. If the cell density is too high, increase the volume of lysis buffer.
Volume should be adjusted to 250–300 µL. [Note]: Insufficient lysis buffer may result in incomplete cell lysis, thereby compromising the quality of protein extraction.
4. Blow into the vial several times and shake vigorously, then place it on ice for 10 minutes, shaking once every 2 minutes.
5. Centrifuge at 12,000 g and 4°C for 5 min.
6. Collect the supernatant.
Tissue sample
1. Cut the tissue into small fragments.
2. Add an appropriate volume of lysis buffer, along with a protease inhibitor cocktail (100×, EDTA-free) (Catalog No.: AP02L084) or PMSF (final concentration of…).
1 mM, but PMSF is insufficient to inhibit all protease activities).
3. Add lysis buffer at a ratio of 1 mL per 100 mg of tissue (if lysis is insufficient, the amount of lysis buffer may be increased as needed; if required…
Increasing the protein concentration can appropriately reduce the volume of lysis buffer used.
4. Homogenize using a homogenizer until complete lysis is achieved (perform this on ice to prevent protein degradation).
5. Centrifuge at 12,000 g and 4°C for 5 min.
6. Collect the supernatant.
Precautions
1. This product is intended solely for scientific research and must not be used for clinical diagnosis, treatment, or any other such purposes.
2. If you need to detect phosphorylated proteins, you must additionally add Phosphatase Inhibitor Cocktail II (50×) (Catalog No.: AP03L025).
3. A small, transparent gel-like precipitate often appears in the lysate prepared with RIPA lysis buffer; this is a normal phenomenon. This transparent gel-like material consists of genomic DNA.
Complexes of interest. In the absence of detecting proteins that bind very tightly to genomic DNA, the supernatant can be directly collected by centrifugation for downstream applications;
If it is necessary to detect proteins that are tightly associated with the genome, the transparent gel-like material can be disrupted and dispersed by sonication, followed by centrifugation.
The supernatant is collected for subsequent experiments. When detecting common transcription factors such as NF-κB, p53, and others, it is usually not necessary to perform ultra…
With sound processing, these transcription factors can be detected.
4. RIPA lysis buffer (medium) contains a high concentration of detergent and is therefore unsuitable for protein quantification by the Bradford assay.
5. If the tissue sample is very small, it can be gently cut into smaller pieces and directly added to the lysis buffer for lysis; vigorous vortexing will ensure thorough cell disruption.
Then, centrifuge again to collect the supernatant for subsequent experiments. The advantage of direct lysis is its convenience, as it eliminates the need for a homogenizer; however, its disadvantage is that it is not as effective as…
Lysis is more thorough, similar to that achieved with a homogenizer.
6. Typically, 150 μL of lysis buffer per well in a 6-well plate is sufficient; however, if the cell density is very high, you may increase the volume of lysis buffer accordingly.
Up to 200 μL or 250 μL.

Table 1: Amount of RIPA Lysis Buffer Used

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This product is intended solely for scientific research and must not be used for clinical diagnosis, treatment, or any other purposes.