Plus RIPA Lysis Buffer

Plus RIPA Lysis Buffer reagent is a complete cell lysis reagent popularly used for cultured mammalian cells. Plus RIPA lysis buffer is highly compatible with immunoassays, protein purification procedures, immunoprecipitation, and western blotting. Cited in 1 publication(s).

Product Info Summary

SKU AR0102-100
Product name Plus RIPA Lysis Buffer
Pack size 100ml
Form Solution (liquid)
Applications Western blotting, ELISA, immunofluorescence, Immunoprecipitation; protein purification; SDS-PAGE; protein assays; epitope tagging; reporter gene analysis

Overview

Form Supplied Ready-to-use 1X solution
Physical State Liquid
Pack Size 100 mL
Content 50mM Tris•HCl pH 7.6, 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 5.0x106 cells in suspension
0.5 mL Plus RIPA Lysis Buffer per 5.0x106 adherent mammalian cells
Storage & Expiration Upon receipt store at 4°C. Plus RIPA Lysis Buffer is stable for one year. Product is shipped on ice.
Assays per kit 200 assays for 5.0x106 cells
100 assays for 0.1g tissue
Compatibility with reagents Fully compatible with Broad Spectrum Protease Inhibitor Cocktail and Broad Spectrum Phosphatase Inhibitor Cocktail
Equivalent Thermofisher (Product No. 89900, 89901), Millipore Sigma (Product No. R0278)
Reagent Type Western Blotting related reagent; Cell lysis buffer; Universal tissue extraction buffer; Detergent solution
Usage Extraction of cytoplasmic, membrane and nuclear proteins
Cite This Product Plus RIPA Lysis Buffer (Boster Biological Technology, Pleasanton CA, USA, Catalog # AR0102-100)

Description

Plus RIPA Lysis Buffer is an enhanced complete cell lysis solution reagent used for rapid and efficient total cell lysis and solubilization of proteins from both adherent and suspension cultured mammalian cells, effectively extracting cytoplasmic, nuclear and membrane proteins. It is more suitable for extracting membrane proteins compared to RIPA Lysis Buffer (Product No. AR0105-100). Protein lysis can be finished in 60 minutes.

Background

RIPA lysis extraction buffer contains non-ionic and ionic detergents which are able to extract protein from wide variety of cell types and membrane structures. RIPA buffer ensures efficient cell lysis and protein solubilization preventing protein degradation and interference with protein immunoreactivity and biological activity. Since most antibodies and protein antigens are not adversely affected by the components of this solution, RIPA buffer-conducted protein extraction is compatible with various downstream immunoprecipitation and molecular pull-down assays, including reporter assays, protein assays, immunoassays and protein purification. RIPA buffer reagent minimizes non-specific protein-binding interactions to keep background low, while allowing most specific interactions to occur, enabling studies of relevant protein-protein interactions.

Important Product Information

• If desired, add protease inhibitor (Product No. AR1182) and phosphatase inhibitor (Product No. AR1183) 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.

Additional Materials Required

• Protease inhibitor (Product No. AR1182) and phosphatase inhibitor (Product No. AR1183)

• 2 ml microcentrifuge tubes

• Tissue homogenizer

• Microcentrifuge capable of spinning at 10,000 x g

• Cell scraper

Procedure for Lysis of Monolayer-cultured Adherent Mammalian Cells

Note: Pre-chill an appropriate volume of Plus RIPA Lysis Buffer at 4°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 0.5 mL 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 areaVolume of the lysis buffer
100mm500-1000μL
60mm250-500μL
6-well plate200-400μL per well
24-well plate100-200μL per well
96-well plate50-100μL per well

4. Centrifuge samples at 14000xg for 10 minutes.

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

Procedure for Lysis of Suspension-cultured Mammalian Cells

Note: Pre-chill an appropriate volume of Plus RIPA Lysis Buffer at 4°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 0.5 mL 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.

Procedure for Lysis of Tissues

Note: Pre-chill an appropriate volume of Plus RIPA Lysis Buffer at 4°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 10 mL 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.

Precautions

• All steps of protein lysis should be operated on ice or at 4°C.

• Use BCA Protein Assay kit (Product No. AR0146) 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.

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AR0102-100 has been cited in 1 publications:

*The publications in this section are manually curated by our staff scientists. They may differ from Bioz's machine gathered results. Both are accurate. If you find a publication citing this product but is missing from this list, please let us know we will issue you a thank-you coupon.

Chen P,Duan Y,Lu X,Chen L,Zhang W,Wang H,Hu R,Liu S.RB1CC1 functions as a tumor-suppressing gene in renal cell carcinoma via suppression of PYK2 activity and disruption of TAZ-mediated PDL1 transcription activation.Cancer Immunol Immunother.2021 Apr 10.doi:10.1007/s00262-021-02913-8.Epub ahead of print.PMID:33837850.
Species: Human,Mouse
AR0102-100 usage in article: APP:WB, SAMPLE:RCC CELLS, DILUTION:NA

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2 Customer Q&As for Plus RIPA Lysis Buffer

Question

Has AR0102-100 been applied on proteins with 12 transmembrane before?

Verified customer

Asked: 2021-01-28

Answer

Our lab hasn't used the Plus RIPA Lysis Buffer (AR0102-100) on proteins with 12 transmembrane. It might be unsuitable for the specific protein. We suggest to look for a specific membrane protein extraction buffer.

Boster Scientific Support

Answered: 2021-02-01

Question

What buffer is recommended for membrane protein extraction?

Verified customer

Asked: 2021-01-24

Answer

For membrane protein extraction, Plus RIPA Lysis Buffer (AR0102-100) is recommended to be used.

Boster Scientific Support

Answered: 2021-01-25

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