Separation method of proteins

A separation method and protein technology, which is applied in the field of protein separation and analysis, can solve the problems of less research and development of stationary phases, and achieve the effects of high column efficiency, wide application range, and simple operation

Inactive Publication Date: 2018-06-29
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Abstract
  • Description
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Embodiment 1

[0032] Take insulin (1), isoelectric point 5.3; cytochrome C (2), isoelectric point 10.7; lysozyme (3), isoelectric point 11.0; transferrin (4), isoelectric point 5.3; bovine serum albumin (5), the standard substance of isoelectric point 4.8, be dissolved in the trifluoroacetic acid aqueous solution of 0.1% volume concentration respectively, prepare the solution that concentration is 5mg / mL, take every kind of protein solution certain amount and mix and be mixed with protein mixed solution, carry out The sample volume is 5 μL. The non-polar group of the stationary phase is n-octane, and the structure of the polar group is as follows:

[0033]

[0034] The column specification is 4.6mm×150mm I.D., 5μm, pore size

[0035] The chromatographic separation conditions on the stationary phase are as follows: column temperature 30°C, flow rate 1mL / min; mobile phase aqueous phase solution is 0.1% volume concentration of trifluoroacetic acid aqueous solution, organic phase is 0.1% ...

Embodiment 2

[0040]Standards of carbonic anhydrase (1), isoelectric point 5.9 and myoglobin (2), isoelectric point 7.0 were respectively dissolved in 10mM ammonium formate aqueous solution, the concentration was 5mg / mL, and the injection volume was 5μL; the structure and implementation of chromatographic packing The same as Example 1, the column specification is 4.6mm×150mm I.D., 5μm, pore size The column temperature is 30°C, the flow rate is 1mL / min; the mobile phase A is ammonium formate aqueous solution, the pH value is 3.0, and the isocratic 10mM; the B phase is acetonitrile, and the B phase gradient is 10%-60% volume concentration, 0-20min; Detector, detection wavelength 280nm;

Embodiment 3

[0044] Recombinant human growth hormone (r-hGH) was dissolved in pure water to prepare a mother solution (prototype) with a concentration of 2 mg / mL, and 100 μL of the mother solution was added to 10 μL of 100 mM dithiothreitol and incubated at 30 ° C for 60 minutes to obtain reduced r-hGH (reduced form), take another 100 μL mother solution and add 5 μL hydrogen peroxide (30% v / v) to incubate at 30°C for 60 minutes, then add an appropriate amount of methionine to terminate the reaction to obtain oxidized r-hGH (oxidized form), injection volume 5 μL . The chromatographic packing structure is the same as in Example 1, and the chromatographic column specification is 4.6mm×150mmI.D., 5μm, and the aperture Column temperature is 30°C, flow rate is 1mL / min; mobile phase A is ammonium formate aqueous solution, pH value is 3.0, isocratic 50mM; B phase is acetonitrile, gradient condition is B phase volume concentration 20%-75%, 0-30min, The pH value is 3.0; UV detector, detection wave...

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Abstract

The invention provides a separation method of proteins. A reversed phase/ion exchange mixed mode chromatography filler adopts a polar copolymerization bonding method and is adopted in the separation process of the separation method, a non-polar group and a polar group are simultaneously bonded on the silica gel surface, a separation mode is reversed phase/electrostatic repulsion interaction, and the separation method uses a reversed phase separation system; the reversed phase/electrostatic repulsion interaction means that 1) a non-polar group on the fixed phase surface provides reversed phasehydrophobic interaction; 2) a pH value of eluent is regulated from acid to neutral, so that a polar group on the fixed phase surface is positively charged; the pH value of the eluent is set to be lessthan an isoelectric point of each of the proteins required to be separated, so that the protein is positively charged; the positively-charged protein and the positively-charged polar group of a fixedphase filler generate electrostatic repulsive interaction, and the various proteins are separated due to different repulsive forces. The separation method provided by the invention has the characteristics of stability, high efficiency and unique selectivity.

Description

technical field [0001] The invention belongs to the technical field of protein separation and analysis. Background technique [0002] Protein is an important part of various biological organisms, and the study of protein is of great significance to the fields of biology, medicine and food. Effective separation analysis is the prerequisite for further in-depth study of proteins. At present, reversed-phase chromatography is one of the effective methods for separating and analyzing proteins. Silica-based alkyl-bonded stationary phase is the most commonly used separation material for reversed-phase chromatography to separate proteins. However, this type of stationary phase can only provide a single hydrophobic force and cannot provide sufficient separation selectivity for complex protein samples. Therefore, it is of great significance to develop a new type of reversed-phase immobilization with specific selectivity for protein separation. [0003] At present, it is an importan...

Claims

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Application Information

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IPC IPC(8): C07K1/20C07K1/18C07K1/16
CPCC07K1/16C07K1/18C07K1/20
Inventor 梁鑫淼丁玲郭志谋胡卓闫竞宇
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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