RIPA （ RIPA lysis Buffer）

RIPA Buffer

Introduction 

The RIPA buffer is one of the most reliable buffers used to lyse cultured mammalian cells from both plated cells and cells pelleted from suspension cultures. This buffer enables protein extraction from cytoplasmic, membrane and nuclear proteins and is compatible with many applications, including reporter assays, protein assays, immunoassays and protein purification. 

Important Product Information  

• RIPA Buffer does not contain protease or phosphatase inhibitors. If desired, add protease inhibitors to the reagent to prevent proteolysis and maintain phosphorylation status of proteins. Add protease and phosphatase inhibitors immediately before use. 

• Use 1mL of cold RIPA Buffer for every 5 × 106 of HeLa or A431 cells (~20µL of packed cells, which is equivalent to ~40mg of cells). To obtain concentrated protein extracts, directly lyse cells on plate and use less buffer.  

• Some protein kinases and other enzymes may be sensitive to the components of the RIPA Buffer, resulting in their decreased activity. In such cases, prepare a RIPA buffer that does not contain sodium deoxycholate and SDS. 

• RIPA Buffer is compatible with the BCA Protein Assay Kit 

 Procedure for Lysis of Monolayer -cultured Mammalian Cells

Note: If desired, add protease and phosphatase inhibitors to the RIPA Buffer immediately before use.

1. Carefully remove (decant) culture medium from adherent cells.

2. Wash cells twice with cold PBS.

3. Add cold RIPA Buffer to the cells. Use 1mL of buffer per 75cm2flask containing 5 × 106 HeLa or A431 cells. Keep on ice for 5 minutes, swirling the plate occasionally for uniform spreading. 

4. Gather the lysate to one side using a cell scraper, collect the lysate and transfer to a microcentrifuge tube. Centrifuge samples at ~14,000 × g  for 15 minutes to pellet the cell debris.   Note: To increase yields, sonicate the pellet for 30 seconds with 50% pulse.  

5. Transfer supernatant to a new tube for further analysis. 

Procedure for Lysis of Suspension-cultured Mammalian Cells

Note: If desired, add protease and phosphatase inhibitors to the RIPA Buffer immediately before use.

1. Pellet the cells by centrifugation at 2500 × g for 5 m inutes. Discard the supernatant. 

2. Wash cells twice in cold PBS. Pellet cells by centrifugation at 2500 × g for 5 minutes. 

3. Add RIPA Buffer to the cell pellet. Use 1mL of RIPA buffer for 40mg (~5 × 106 of HeLa cells) of wet cell pellet. Pipette the mixture up   and down to suspend the pellet.   Note: To increase yields, sonicate the pellet for 30 seconds with 50% pulse.  

4. Shake mixture gently for 15 minutes on ice. Centrifuge mixture at ~14,000 × g for 15 minutes to pellet the cell debris. 

5. Transfer supernatant to a new tube for further analysis. 

Procedure for Tissue Lysis

1. If desired, add protease inhibitors to the RIPA Buffer just before use.  

2. Weigh tissue samples. Use a ratio of ~1 g of tissue to 20mL RIPA Buffer. Use a smaller volume of RIPA Buffer if a more concentrated protein extract is required. 

3. Add the appropriate amount of RIPA Buffer to the tissue sample and homogenize.

4. Pipette the mixture to a new tube, Shake mixture gently for 15 minutes on ice.

5. Centrifuge the sample at 14,000 × g for 15 minutes to pellet cell/tissue debris.  

5. Collect supernatant and continue with downstream analysis or further purification. 

Origin:  Pioneer Biotechnolgy,  Inc of China

Distributor: Pioneer Biotechnolgy,Inc

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