Introduction:

10X RIPA protein extraction reagent is designed for extracting whole intact proteins from any type of mammalian tissues/cells. The ready-to-used optimal formula can help you to deal with any type of tissue/cell samples more easily and getting more protein content than other competitor brands. In addition, the optimized protease inhibitor and phosphatase inhibitor are incorporated into 10X RIPA protein extraction reagent to protect your lysate sample from degradation.

Protocol:

A. RIPA preparation

1. Mix well by end-over-end at least 5 times before use.

2. Dilute 10X RIPA Buffer to a 1X solution using ultrapure water.

B. cell sample extraction

1. Mix well by end-over-end at least 5 times before use.

2. Dilute 10X RIPA protein extraction reagent to a 1X solution using ultrapure water. In average, please use 1ml 10X RIPA protein extraction reagent to lysis 5x107 to 1x108 cells.More cells can be added if the protein contents are low in your target cell type.

3. Lyse the cells with extraction buffer, on ice, with vortexing at 2 minutes intervals.

4. Centrifuge samples at 10000 x g for 5 minutes to precipitate pellets.

5. The supernatant are protein containing extract, collect it and performing the downstream analysis.

C. tissue protein extraction

1. In average, please use 20ml 10X RIPA protein extraction reagent to lysis 1g tissues. More tissues can be added if the tissue protein contents are low; or more RIPA buffer are added if the lipid portion are too much in the samples. Remember always put the samples on ice during homogenization.

2. After RIPA step by tissue homogenizer, centrifuge samples at 10000 x g for 5 minutes to precipitate pellets.

3. The supernatant are protein containing extract, collect it and performing the downstream analysis.

Caution:

1. Please avoid mixing with strong acid or basic solutions.

2. Please wear gloves and goggles to avoid skin-contact before operation.

Storage:

It can be storage at 4°C. For long storage, please freeze below -20°C.