Method for preparing immobilized metal ion affinity chromatographic monolithic column

An immobilized metal and monolithic column technology is applied in the field of preparation of immobilized metal ion affinity chromatography materials, can solve problems such as loss, and achieve the effects of low cost, strong acid and alkali resistance, and good selectivity

Active Publication Date: 2012-11-07
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Application Information

AI Technical Summary

Problems solved by technology

In traditional immobilized metal ion affinity chromatography materials, each metal ion is only coordinated with one IDA or NTA ligand, so it is easy to lose during loading or elution
The commonly used metal ions, such as Fe 3+ , Ga 3+ 、Ni 2+ etc. can also bind carboxyl groups, resulting in non-specific adsorption of acidic or histidine-containing peptides

Method used

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  • Method for preparing immobilized metal ion affinity chromatographic monolithic column
  • Method for preparing immobilized metal ion affinity chromatographic monolithic column
  • Method for preparing immobilized metal ion affinity chromatographic monolithic column

Examples

Experimental program
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Effect test

Embodiment 1

[0025] Follow the steps below to prepare an immobilized metal ion affinity chromatography column.

[0026] 1) Modification of chelating groups: Add 2 mg / mL AMPA and 5 mg / mL cyano-sodium borohydride NaCNBH 3 100 mM phosphate buffer (pH 8.0) was pumped into the column and reacted at room temperature for 6 hours. The monolithic column carrier with surface-modified phosphoric acid chelating groups can be prepared. The specific parameters of the synthesis example are shown in Table 1 below.

[0027] 2) Immobilization of metal ions: containing 20-100mM Ti(SO 4 ) 2 The aqueous solution is reacted at room temperature for 6-24 hours; the prepared monolithic column is washed with water for 0.5-6 hours. That is, the immobilized metal ion affinity chromatography column was prepared (the specific parameters are shown in Table 1 below).

[0028] Table 1. Modification of chelating groups on the surface of monolithic materials and immobilization parameters of metal ions.

[0029] ...

Embodiment 2

[0035] The difference from Example 1 is that step 3) adopts ZrOCl in the immobilization process of metal ions 2 Aqueous solution of Ti(SO 4 ) 2 of aqueous solution. The specific parameters of the synthetic examples are shown in Table 1.

[0036] The advantages of the present invention are: good selectivity to phosphorylated peptides, reusable, large enrichment capacity, high recovery rate, simple preparation method, good permeability, low back pressure, good mechanical strength, Strong acid and alkali resistance.

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Abstract

The invention relates to preparation of an immobilized metal ion affinity chromatographic material, and particularly relates to a method for preparing an immobilized metal affinity ion chromatographic monolithic column. In the method, a monolithic column material is prepared by using an organic-inorganic hybrid silica gel monolithic material of which the surface carries aldehyde groups as a vector; a phosphate buffer solution containing aminomethylphosphonic acid and sodium cyanotrihydroborate NaCNBH3 flows through the monolithic column material continuously to prepare a monolithic material vector of which the surface carries aldehyde chelation groups; and an aqueous solution of transition metal ions flows through the material vector to prepare the immobilized metal ion affinity chromatographic monolithic column. High-selectivity enrichment of phosphorylated peptide in a hybrid peptide section can be realized by utilizing the immobilized metal ion affinity chromatographic monolithic column. The invention has the advantages that the monolithic column has good selectivity to the phosphorylated peptide, can be recycled, and has the characteristics of large enrichment capability and high recovery rate; and the preparation method has the characteristics of simplicity, convenience, good permeability and small back pressure, and the prepared immobilized metal ion affinity chromatographic monolithic column has good mechanical strength and strong acid and alkali resistance.

Description

technical field [0001] The present invention relates to the preparation of immobilized metal ion affinity chromatographic materials, specifically a phosphoric acid chelation immobilized metal ion affinity chromatographic column based on organic-inorganic silica gel hybrid matrix, which is applied to the high phosphorylated peptide Selective enrichment. Background technique [0002] Immobilized metal ion affinity chromatography is a technique for selectively enriching phosphorylated peptides based on the affinity between immobilized metal ions and the phosphate groups of phosphorylated peptides. Compared with other phosphorylated peptide enrichment methods, immobilized metal ion affinity chromatography is the most widely used, and can enrich various phosphorylated amino acid residues. The development of immobilized metal ion affinity chromatography has been widely concerned by many researchers (Paradela, A.; Albar, J.P.J. Proteome Res. 2007, 7, 1809-1818 & Han, G.H; Ye, M.L....

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): B01J20/281B01D15/22G01N30/60C07K1/16
Inventor 张丽华侯春彦马俊锋梁振张维冰张玉奎
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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