When it comes to protein extraction, everyone will inevitably think of RIPA lysis buffer. The lysis buffer can be added to the sample and centrifuged to complete protein extraction. However, did we really use RIPA lysis buffer correctly? RIPA lysis buffer is a commonly used lysis buffer for extracting proteins from animal cells or tissues. Its working principle is to use detergents (surfactants) to lyse cell membranes and nuclear membranes, thereby obtaining RIPA soluble proteins in cells and tissues.
Selection of RIPA cracking solution
RIPA cracking solution mainly contains detergents, buffer systems, isotonic systems, denaturing agents, stabilizers, etc. Among them, detergent is an important component in cell lysate, which is a zwitterionic molecule composed of hydrophobic tail end groups and polar hydrophilic head end groups. They can reduce the surface tension of water, so they are also called surfactants, which play a role in destroying lipid bilayers, rupturing cells, and dissolving proteins.
Types of cleaning agents
Different types of detergents have different efficiency in sample lysis. RIPA lysis buffer has multiple formulations, usually using different combinations and concentrations of detergents. Therefore, the amount and types of proteins that can be dissolved in RIPA lysis buffer with different formulations will vary greatly, and the obtained protein profiles will also differ, resulting in different detection results in subsequent experiments. It is necessary to select a suitable RIPA cracking solution formula based on experimental samples and downstream experiments.
Precautions for using ripa cracking solution
Due to the mild lysis intensity of most RIPA lysates, they are more effective in extracting soluble proteins in the cytoplasm and some proteins highly expressed on the cell membrane and nucleus. Therefore, RIPA lysates are suitable for the extraction of such proteins and are recommended for qualitative detection in WB experiments.
When extracting protein from RIPA lysate, two components are produced after centrifugation, namely RIPA soluble component (supernatant) and RIPA insoluble component (white precipitate), which is normal. Try to avoid settling and absorbing the supernatant for downstream experiments.
If the sample becomes viscous after adding RIPA lysis buffer, it indicates that the sample has been too thoroughly lysed to remove nucleic acid residues. In this case, a lower strength lysis buffer formula should be used, and ultrasound treatment should be added or the total protein extraction kit (Li Ji Biological Cat: AP01L204) should be replaced. Column method can use a centrifugal column to intercept nucleic acid residues and solve the problem of sample viscosity. There are no insoluble components, and a more complete protein profile can be obtained.
When using RIPA lysis buffer to extract tissue samples, it is challenging to efficiently extract tissue proteins from different samples due to the significant differences in tissue samples. For example, fibrous tissue and collagen tissue (such as heart and kidney) and soft tissue (such as spleen and liver) need to be matched with different grinding schemes to obtain better results.
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