Method for separating copper proteome by using copper-chelated magnetic beads

Abstract

The invention discloses a method for separating copper proteome by using copper-chelated magnetic beads, and relates to the technical field of protein chemistry. The method comprises the following steps: taking a biological tissue or cell, after fully grinding with liquid nitrogen, adding protein extracting liquid, and after uniformly mixing, carrying out low-temperature centrifugation, so as to obtain a denatured protein solution; mixing the copper-chelated magnetic beads with the denatured protein solution, and incubating to obtain a magnetic bead protein mixed solution; adding a buffer solution 1 with the volume 1 time of that of the magnetic bead protein mixed solution into the magnetic bead protein mixed solution for suspension, pouring half of the supernate under the external magnetic field, and repeating for 4-5 times to obtain a mixed solution A; adding a buffer solution 2 with the volume 1 time of that of the mixed solution A into the mixed solution A, separating all the specific copper-binding proteins, namely, the copper proteome, and collecting the copper proteome under the external magnetic field. According to the method, the metalloproteome is separated under a denaturing condition by using magnetic bead-IDA, so that the procedures are greatly simplified, and the obtaining efficiency of metalloproteins is improved; a method suitable for separating the metalloproteome from the tissue or cell under the excess heavy metal treatment condition is established.

Description

technical field [0001] The invention relates to the technical field of protein chemistry, in particular to a method for separating copper proteomes by using copper chelating magnetic beads. Background technique [0002] The National Pollution Survey Bulletin shows that the total rate of soil pollution in the country is 16.1%, of which heavy metal pollution accounts for more than 80%. Most of the heavy metals in the soil are easy to accumulate in plants and endanger human health through the food chain. On the other hand, because the cysteine, methionine, and histidine residues of proteins have high affinity for divalent metal ions, excessive heavy metals in cells may interfere with the binding of essential metal elements to proteins, Cause protein denaturation or enzyme inactivation, interfere with normal metabolic process. [0003] Proteomics technology has become an important research method in the field of functional genomics. All proteins with metal binding sites in a ...

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Problems solved by technology

However, the disadvantage of this method is that NTA is very toxic, and the price is not cheap, and the scope of application is limited.

[0006] Since the metal-binding sites of most proteins in the natural state exist inside the molecule, it is difficult for us to isolate the complete metalloprotein group by immobilizing the metal, but this has not been addressed in the previous research literature on metal-binding proteins. consider

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