Understand the use and preparation of buffers such as TRIS-HCL - complete WB experiments quickly
Solubilization and stabilization of proteins
To ensure the success of electrophoresis when doing WB, the first thing to do is to destroy the aggregation between proteins. Ideally, both cell lysis and protein solubilization are performed in the electrophoresis sample buffer. If there is no way to do this, prepare the protein in a solubilization solution, which contains a descaling agent, a denaturing/reducing agent, and a buffer that is compatible with electrophoresis. The lysis buffer contains various detergents that can help destroy cells and release proteins, making them soluble in solution. However, different types of proteins react differently to buffers and detergents.
The cell extract that took a long time to prepare could not detect the target protein. After the WB experiment was finally completed, it was found that the band was weak or no band. This is the current situation of WB for scientific researchers. If the target protein is not dissolved in solution, or the protein-protein interaction is special, you need to try other special buffers and exchange to remove the descaling agent. So what are the principles to follow on the selection and use of buffers and reducing agents when performing WB?
General principles of buffer use
Type of protein Commonly used buffers Precautions
Whole cell lysates and membrane-bound proteins RIPA, NP-40 Strong properties of RIPA buffer for poorly soluble proteins
Nuclear/mitochondrial protein selection RIPA typically begins with a fractionation protocol to increase the concentration of specific organelles and proteins of interest.
Cytoplasmic proteins Tris-HCl, RIPA (Tris-HCl is sometimes more advantageous) The conditions need to be determined according to the experiment to obtain more target proteins.
Native state proteins CHAPS (zwitterionic descaling agents that better preserve proteins in their native state) are commonly used in isoelectric focusing (IEF) and 2-D electrophoresis.
Common lysis buffer formulations
In order to help you better understand the principles of using buffers when conducting WB experiments, here are some commonly used buffer formulations and commonly used reducing agents. The lysis buffer formulations are as follows:
Tris-HCl formula: 20mM Tris-HCl, pH7.5
CHAPS formula: 150mM KCI; 50mM HEPES (4-hydroxyethylpiperazineethanesulfonic acid); 0.1% CHAPS (3-3-cholamidopropyldimethylamino-propanesulfonic acid inner salt)
RIPA formulation: 150mM NaCl; 1% NP-40 or Triton X-100; 0.5% sodium deoxycholate; 0.1% SDS; 50mM Tris (pH8.0)
NP-40 formula: 150mM NaCl; 1% NP-40 or Triton X-100; 50mM Tris (pH 8.0)
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