Plus RIPA Lysis Buffer

Liquid. 50mM Tris-HCl pH7.6, 150mM NaCl, 1% TritonX-100, 1% sodium deoxycholate, 0.1% SDS

Recommended working concentration:
10 mL Plus RIPA Lysis Buffer per gram of tissue
0.5 mL Plus RIPA Lysis Buffer per 5.0x10^6 cells in suspension
0.5 mL Plus RIPA Lysis Buffer per 5.0x10^6 adherent mammalian cells (lot specific)

Upon receipt store at 4 degree C. Plus RIPA Lysis Buffer is stable for one year. Product is shipped on ice.

Fully compatible with Broad Spectrum Protease Inhibitor Cocktail and Broad Spectrum Phosphatase Inhibitor Cocktail

Western Blotting related reagent; Cell lysis buffer; Universal tissue extraction buffer; Detergent solution

Extraction of cytoplasmic, membrane and nuclear proteins

If desired, add protease inhibitor and phosphatase inhibitor to the lysis buffer to prevent proteolysis and maintain phosphorylation status of proteins.
Some protein kinases and other enzymes may be sensitive to the components of the Plus RIPA Lysis Buffer, resulting in their decreased activity. In such cases, prepare a Plus RIPA Lysis Buffer that does not contain sodium deoxycholate and SDS.

Protease inhibitor and phosphatase inhibitor
2mL microcentrifuge tubes
Tissue homogenizer
Microcentrifuge capable of spinning at 10,000 x g
Cell scraper

Note: Pre-chill an appropriate volume of Plus RIPA Lysis Buffer at 4 degree C. If desired, add protease inhibitor and phosphatase inhibitor to the lysis buffer immediately before use.
1. In a microcentrifuge tube, resuspend 5×10⁶ cells in the growth media by scraping the cells off the surface of the plate with a cell scraper. Centrifuge harvested cell suspension at 600xg for 5min, then carefully remove and discard the supernatant.
2. Resuspend the cells in chilled PBS. Centrifuge at 600xg for 5min, then carefully remove and discard the supernatant.
3. Add 0. 5mL of chilled Plus RIPA lysis buffer to the cell pellet. Vortex briefly. Incubate on ice for 30 minutes.
4. Centrifuge samples at 14000xg for 10 minutes.
5. Transfer supernatant to a new tube for further analysis.
Note: Plus RIPA lysis buffer can be added directly to the flask containing cells. Please see the following procedures.
1. Carefully remove culture medium from adherent cells.
2. Wash cells with chilled PBS. Carefully remove PBS.
3. Add chilled Plus RIPA lysis buffer to the cells. Vortex briefly. Incubate on ice for 30 minutes. (For the volume of the lysis buffer, follow the instructions listed below)
SIZE of the plate/surface area: Volume of the lysis buffer
100mm: 500-1000uL
60mm: 250-500uL
6-well plate: 200-400uL per well
24-well plate: 100-200uL per well
96-well plate: 50-100uL per well
4. Centrifuge samples at 14000xg for 10 minutes.
5. Transfer supernatant to a new tube for further analysis.

Note: Pre-chill an appropriate volume of Plus RIPA Lysis Buffer at 4 degree C. If desired, add protease inhibitor and phosphatase inhibitor to the lysis buffer immediately before use.
1. In a microcentrifuge tube, harvest 5×10⁶ cells by centrifugation at 600xg for 5min. Carefully remove and discard the supernatant.
2. Resuspend the cells in chilled PBS. Centrifuge at 600xg for 5min, then carefully remove and discard the supernatant.
3. Add 0.5mL of chilled Plus RIPA lysis buffer to the cell pellet. Vortex briefly. Incubate on ice for 30 minutes.
4. Centrifuge samples at 14000xg for 10 minutes.
5. Transfer supernatant to a new tube for further analysis.

Note: Pre-chill an appropriate volume of Plus RIPA Lysis Buffer at 4 degree C. If desired, add protease inhibitor and phosphatase inhibitor to the lysis buffer immediately before use.
1. Place the fresh tissue into chilled PBS and rinse several times. Mince the tissue into small pieces.
2. Add Plus RIPA Lysis Buffer to the tissue at 10:1. (i. e.. Add 10mL chilled lysis buffer per gram of tissue. ) Use a smaller volume of reagent if a more concentrated protein extract is required.
3. Homogenize for several minutes at high speed until no tissue chunks remain.
4. Incubate on ice for 30 minutes.
5. Centrifuge at ~10000 x g for 10 minutes.
6. Transfer supernatant to a new tube for further analysis.

All steps of protein lysis should be operated on ice or at 4 degree C.
Use BCA Protein Assay kit to quantify lysed proteins. Bradford Protein Assay kit is not recommended.
There might be some transparent gel complex containing genomic DNA in lysed proteins. The protein fractions can be used for further analysis after centrifugation if target proteins have little connection with genomic DNA. When detecting target proteins related closely to genomic DNA, sonicate gel complex and then centrifuge to collect supernatant for further analysis. Common transcription factors such as NFKB, p53 can be detected without sonication.

Assistive Reagent

Manufactured in an ISO 13485:2003 and EN ISO 13485:2012 Certified Laboratory.