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A complex collection method based on capillary zone electrophoresis at low pH

A technology of capillary electrophoresis and zone electrophoresis, applied in separation methods, chemical instruments and methods, and separation of dispersed particles, can solve the problems of cumbersome collection process and small collection of complexes, and achieve the purpose of suppressing electroosmotic flow and increasing online enrichment The effect of concentration and inhibition of adsorption

Inactive Publication Date: 2016-01-20
BEIJING INSTITUTE OF TECHNOLOGYGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In the presence of electroosmotic flow, the collection of complexes in CE-SELEX technology needs to determine the collection time window, and usually requires multiple analyzes to collect a large number of complexes, and the collection process is cumbersome
Therefore, it is necessary to provide a method for collecting large-volume complexes in a single analysis, so as to overcome the shortcoming of the small amount of complexes collected during the screening process.

Method used

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  • A complex collection method based on capillary zone electrophoresis at low pH
  • A complex collection method based on capillary zone electrophoresis at low pH
  • A complex collection method based on capillary zone electrophoresis at low pH

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Preparation of original Trf stock solution: Weigh 0.0040 g of Trf, add 500 μL of pure water to prepare 1.0×10 -4 mol / L original Trf stock solution, stored at 4°C.

[0033] The original Trf stock solution was diluted with pure water to obtain a Trf sample solution, and 10 μL of the concentration was 5.0×10 -6 The Trf sample solution of mol / L, in Agilent7100 capillary electrophoresis instrument, through 50mbar pressure injection 10s, with 50mM, the NaH of pH value is 2.6 2 PO 4 -H 3 PO 4 The solution is electrophoresis running buffer, and low pH capillary zone electrophoresis is carried out. The electrophoresis analysis time is 20min, the analysis voltage is +10KV, and the analysis temperature is 15°C. The outlet end of the capillary is the negative electrode, and the inlet end is the positive electrode. The result is as figure 1 shown.

[0034] Depend on figure 1 It can be seen that under positive voltage, Trf produces a signal peak at 9.5 min, indicating that Trf...

Embodiment 2

[0036] The preparation method of the original ssDNA stock solution is as follows: the ssDNA is in the form of crystals, centrifuged at 3000 rpm for 5 minutes, and added with pure water to prepare a 1.0×10 -4 The mol / L original ssDNA stock solution was cooled on ice in a water bath at 94°C for 5 minutes, and stored at 4°C.

[0037] Dilute the original ssDNA stock solution with pure water to obtain ssDNA sample solution, and dilute 10 μL to a concentration of 5.0×10 -6 The mol / L ssDNA sample solution was injected into the Agilent7100 capillary electrophoresis instrument through 50mbar pressure for 10s, and the NaH with 50mM and pH value of 2.6 2 PO 4 -H 3 PO 4 The solution is the electrophoresis running buffer, and the capillary zone electrophoresis with low pH value is carried out. The electrophoresis analysis time is 13min, the analysis voltage is -10KV, the analysis temperature is 15°C, the outlet end of the capillary is the positive electrode, and the inlet end is the neg...

Embodiment 3

[0041] Dilute 5 μL to a concentration of 1.0 x 10 -5 mol / L Trf sample solution with a concentration of 4.0×10 in 5 μL -6 mol / L ssDNA sample solution was mixed to obtain a mixed sample, and incubated on ice for 0.5h.

[0042] The mixed sample was injected into the Agilent7100 capillary electrophoresis instrument through 50mbar pressure for 10s, with 50mM NaH with a pH value of 2.6 2 PO 4 -H 3 PO 4 The solution is an electrophoresis running buffer for low pH capillary zone electrophoresis separation. During the electrophoresis separation process, centrifuge tubes with 30 μL of electrophoresis running buffer are used as collection bottles at both ends of the capillary; the electrophoresis analysis voltage is +10KV, the analysis temperature is 15°C, and the analysis time is 20 minutes. The outlet end of the capillary is negative, and the inlet end is is the positive electrode, the DAD detector results are as follows image 3 shown.

[0043] The result is: Trf has a net posi...

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Abstract

The invention relates to a compound collecting method based on low-pH-value capillary zone electrophresis, and belongs to the technical field of bio-separation and analysis. The method comprises the steps of incubating protein and a fluorescence labeling ssDNA (single-stranded DNA) library on ice after mixing; separating an incubated mixed sample by low-pH-value capillary zone electrophresis, wherein the pH value of electrophresis running buffer liquid is less than 3; in electrophresis separation, moving dissociated protein, dissociated ssDNA and a compound formed by the protein and the ssDNA in the mixed sample in the direction opposite to the charge characteristic according to charge characteristics of the dissociated protein, dissociated ssDNA and the compound formed by protein and ssDNA respectively; carrying out CE-LIF (capillary electrophresis laser induced fluorescence detector) analysis on collection collected at the outlet end and the inlet end of a capillary, and testing the method to be feasible. The method can inhibit electroosmotic flow and inhibit the adsorption of positive charge samples and alkaline protein on the inner wall of the capillary; the online separation of samples with different charge types is realized; the sample online enrichment amount can be increased through increasing the sample feed amount; more protein and ssDNA compounds can be obtained through analysis once.

Description

technical field [0001] The invention relates to a method for collecting complexes based on low-pH capillary zone electrophoresis. The complexes are protein and ssDNA complexes, and belong to the technical field of biological separation and analysis. Background technique [0002] The oligonucleotide library is a class containing 10 13 ~10 15 A mixture of single-stranded DNA molecules (single-stranded DNA, ssDNA) with different bases, which may contain one or several DNA molecules that bind to target molecules with high specificity and high affinity. Nucleic acid aptamer (Aptamer) refers to the oligonucleotide with high affinity and high specificity for the target molecule, which is screened from the random oligonucleotide library by the exponential enrichment ligand system evolution (SELEX) technology Acid sequence ligands. Nucleic acid aptamers have high affinity, strong specificity, easy preparation and modification, and wide distribution of target molecules. [0003] C...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): B01D57/02
Inventor 屈锋李倩赵新颖刘弘洋
Owner BEIJING INSTITUTE OF TECHNOLOGYGY
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