The Protein Man's Blog | A Discussion of Protein Research

The RIPA buffer, or Radioimmunoprecipitation assay buffer, is ideal for whole-cell extracts, as well as protein assays. However, it is an essential component in other applications too. 

What is a RIPA Buffer?

The RIPA buffer is a lysis buffer essential for the rapid, efficient cell lysis and solubilization of proteins from both adherent and suspension-cultured mammalian cells. The G-Biosciences' RIPA Lysis & Extraction Buffer is a highly reliable buffer for the lysis of adherent and suspension mammalian cells. It results in the subsequent release of the cytoplasmic, membrane, and nuclear proteins from adherent and suspension-cultured mammalian cells. It is fully compatible with various applications, including reporter assays, protein assays, immunoassays, and other protein purification techniques. The RIPA Lysis & Extraction Buffer is also a ready-to-use solution that requires little to no preparation. It is fully compatible with our selection of individual protease inhibitors and cocktails.

How to Prepare the RIPA Buffer

Standard RIPA Lysis Buffer is 150mM sodium chloride, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 25mM Tris, pH 7.4. The RIPA buffer does not contain protease or phosphatase inhibitors to protect proteins following lysis. To prevent proteolytic breakdown and maintain the protein’s phosphorylation state, you should add protease and phosphatase inhibitor cocktails, such as the G-Biosciences' ProteaseArrest™ and PhosphataseArrest™.

Before use, you should also pre-chill the RIPA Lysis & Extraction Buffer and the added inhibitors.

RIPA Buffer Applications

The RIPA Lysis & Extraction Buffer is fully compatible with many applications, including a Western blot or immunoprecipitation assay. Since it contains three non-ionic and ionic detergents, it is also ideal for protein extraction from cultured cells and cell lysis. In cell lysis, the cell membrane breaks down while the proteins divide from the non-soluble parts of the cell. Depending on the location of the protein of interest, a different lysate buffer obtains a high yield and the purity of the protein. The RIPA buffer is essential to the lysis of adherent cells, suspension cells, and tissue. However, there are specific steps you must follow in every procedure.

The Lysis of Adherent Cells

The Protocol for Lysis of Adherent Cells

• Remove the growth media from the cells and wash the cells twice with ice-cold PBS.
• Add 1ml ice-cold RIPA Lysis & Extraction Buffer to every 75cm² flask containing 5 x 10⁶ mammalian cells.
• Incubate on ice for 5-15 minutes with periodical pipetting.
• Use a cell scraper to pool the cell lysate in the flask and transfer to a microcentrifuge tube.
• Centrifuge at ~14,000g for 15 minutes to pellet cell debris. Sonicate the pellet to increase protein yield for 30 seconds at a 50% pulse.

The Lysis of Suspension Cells

The Protocol for Lysis of Suspension Cells

• Centrifuge the cell suspension at 2,500g for 5 minutes to collect the cells. Discard the supernatant.
• Wash the cells twice in ice-cold PBS, pelleting the cells as before.
• Add 1ml ice-cold RIPA Lysis & Extraction Buffer to every 40mg or ~5 x 10⁶ or ~20μl wet pellet of mammalian cells. Pipette up and down to mix.
• Centrifuge the cell suspension at ~14,000g for 15 minutes to pellet cell debris. Sonicate the pellet to increase protein yield for 30 seconds at a 50% pulse.
• Transfer the supernatant to a fresh tube for downstream applications.

The Lysis for Lysis of Tissue

The Protocol for Lysis of Tissue

• Add 1ml ice-cold Lysis & Extraction Buffer to every 100mg of mammalian tissue.
• Sonicate the tissue on ice with ~5 x 30 seconds at a 50% pulse. Allow the sample to cool between each sonication burst. Ensure that you homogenize the tissue entirely before proceeding.
• Incubate on ice for 5-15 minutes with periodical pipetting.
• Centrifuge the cell suspension at ~14,000g for 15 minutes to pellet cell debris. Transfer the supernatant to a fresh tube for downstream applications.

G-Bio is Your Go-To Destination for Cell Lysis

G-Biosciences offers a variety of products for your cell lysis needs, including our Lysis & Extraction Buffer. Visit our website to claim yours today!