Treatment of insoluble proteins
Leukocyte filter for sale China shares that Proteomics has become a hot topic in the post-genome era and a new breakthrough in the field of life science. In this paper, the general processing methods of protein samples were reviewed in terms of the treatment of proteins with strong hydrophobicity, polar proteins, high-abundance proteins, enrichment of low-abundance proteins, removal of interfering substances from the sample solution, and mass spectrometric pretreatment of different dyes.
1. Treatment of insoluble proteins
The solubility of protein in natural state is generally low, and the solubilization of protein affects the final result and determines the success or failure of the whole experiment. Due to the heterogeneity of proteins and the influence of non-protein impurities, it is still very challenging to dissolve all proteins at the same time. At present, the principle of increasing protein solubility is to add reagents to destroy the internal interaction force of protein polymer, so that the protein polymer is lysed into a single polypeptide with strong solubility. These reagents include dissociation agents (urea and thiourea), detergents (CHAPS and triton-100), reductants (DTT, TBP) and protease inhibitors.
Urea is a neutral liquid release agent. Urea with a concentration of 5-9 mol/L can effectively destroy the secondary structure of the protein, expand the protein, and expose ionizable structure groups to enhance its solubility. It is worth noting that the urea and thiourea at higher temperature can be hydrolyzed into cyanate and thiocyanate respectively, lead to artificially modified protein, so the sample temperature is not more than 37 ℃, carrier ampholytes added with urea buffer solution can remove cyanate. At present, improving the solubility of membrane related proteins that form complex with membrane lipids is the biggest challenge in sample processing in proteomics research, and stain remover is extremely important for the extraction of membrane protein complex. Detergent is divided into ion type (SDS), non-ion type (tritonx-100) and ampholytic ion type (CHAPS and asb-14). The concentration range of detergent is generally 1% to 4%. Ionic detergent has better solubilization effect on hydrophobic protein and membrane protein, but it interferes with non-denaturation electrophoresis and isoelectric focusing. In order not to affect sds-page, it is better to choose zwitterionic and non-ionic detergent.
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