CN104672297A

CN104672297A - Screening method of optimal condition for preparing amino-containing micro-molecular substance and protein conjugate based on EDC reaction

Info

Publication number: CN104672297A
Application number: CN201310654333.1A
Authority: CN
Inventors: 张峰; 王晓伟; 钟睿博
Original assignee: Inner Mongolia Agricultural University
Current assignee: Inner Mongolia Agricultural University
Priority date: 2013-12-03
Filing date: 2013-12-03
Publication date: 2015-06-03

Abstract

The invention provides a screening method of an optimal condition for preparing an amino-containing micro-molecular substance and protein conjugate based on EDC reaction. An experiment process method is designed into a concentration gradient screening method. According to the method, an optimal reaction condition, namely molecular number (the ratio of amount of substance) of ECD, proteins and small molecular substances, for effectively coupling a protein molecule (containing free carboxyl: -COOH) and a small molecular substance (containing primary amine groups: -NH2) by utilizing EDC (1-ethyl-(3-dimethyl amino propyl) carbonyl diimine hydrochloride) can be quickly found, and thus, the method is a convenient, effective and scientific method having a trace amount for screening an optimal experiment condition. In the experiment process, EDC is used for activating carboxyl, is a condensating agent of carboxyl and amino, and is high in extremely-decomposed property; therefore, EDC needs to be prepared once being used instantly and is used as quick as possible after the EDC is prepared; the coupling rate is detected by using a conventional electrophoresis method.

Description

A kind of screening method containing amino small-molecule substance and protein conjugate top condition based on EDC reaction preparation

Technical field:

The present invention relates to the method for attachment of chemical field, this patent describes the screening method containing amino small-molecule substance and protein conjugate top condition based on EDC reaction preparation.

Background technology:

Small-molecule substance is often coupled on a larger protein molecule, is applied in based on fields such as immunoreactive detection, diagnosis and treatment as haptens.Normally used method is Euplotes woodruffi and EDC crosslinking mainly, the former due to joint efficiency low, crosslinked seriously polluted between albumen, and glutaraldehyde itself has an increased the link distance between small molecules and protein, finally make cross-linking agent yardstick become the shortcoming such as large, at present substantially replace by EDC crosslinking.Experimenter is when doing EDC experiment, all ordinary practice is in the molecule proportioning with reference to some forefathers, and the PI value of amino quantity and different proteins contained by different small molecules, the ratio of carboxyl and amino, all to a certain extent there is larger difference, so its best molecule proportioning should be redefined for different protein and micromolecular crosslinking experiments.The EDC method coupling adopted in this patent provides the less optimization reaction method of a kind of quick, efficient, required reaction volume containing amino small-molecule substance and albumen, can be the new top condition filtering out reaction containing amino small-molecule substance and the coupling of albumen phase based on EDC.

Summary of the invention:

The invention provides a kind of efficient, the screening method easily of preparing small molecules and protein molecule optimum reaction condition, the Molecules ratio (ratio of amount of substance) of EDC and protein and small-molecule substance in linked reaction can be determined rapidly.Its core technology is, by EDC, protein and small molecules three by twice descending concentrations serial experiment, then is made up of electrophoresis race glue detection reaction result.

Accompanying drawing illustrates:

The coupling of Fig. 1 EDC method is containing amino small molecules and protein ladder table;

Albumen and containing amino micromolecular Molecules than the Molecules for 1:500, EDC than from No. 1 to No. 11, pipe reduces by half successively.

Fig. 2 SDS-PAGE glue schematic diagram;

These 12 bands are respectively sample in No. 1-12 pipe from right to left, and wherein the 12nd band is control sample.

Embodiment:

1) get row's PCR pipe and numbering 1-12, in these 12 pipes, add 10 μ L deionized waters respectively.

2) the EDC aqueous solution (768mmol/L) that 10 μ L newly prepare is joined in No. 1 pipe, liquid-transfering gun mixes, from No. 1 pipe, extract 10 μ L joins in No. 2 pipes again, and liquid-transfering gun mixes, then extracts 10 μ L and join in No. 3 pipes, extracting 10 μ L after mixing again joins in No. 4 pipes, extracting 10 μ L after mixing again joins in No. 5 pipes, by that analogy, joins No. 11 pipes again until extract 10 μ L after No. 10 pipe mixings, extract 10 μ L after mixing, abandon it.

3) in 1-12 pipe, add 10 μ L protein (6 μm of ol/L) and 10 μ L small molecules (3mmol/L) respectively, react 3 hours under room temperature.

4) conventional electrophoretic method is utilized to detect as above sample, the SDS-PAGE glue of usual employing 10% runs glue 2 hours under 100V voltage, coomassie brilliant blue staining, decolour, take pictures and carry out the delayed contrast of band: the EDC molecule consumption (consumption can cause the crosslinked of protein self too much, and coupling efficiency is too low again very little) that can filter out rapidly coupling top condition.

5) EDC and small molecules sample are exchanged, repeat above 1)-4) process: wherein need 4) in the best EDC amount that finds be applied to above-mentioned experiment, the molecular ratios holding EDC and albumen is constant, convert micromolecular amount of substance (molecule number: pipe reduces by half successively from No. 1 to No. 11, No. 12 pipes are control tube).

In view of pH value and temperature are the key factors affecting reaction yield; the technician of the industry should be appreciated that; the present invention is not restricted to the described embodiments; what describe in above-described embodiment and specification sheets just illustrates principle of the present invention; without departing from the spirit and scope of the present invention; the present invention also has various changes and modifications, and these changes and improvements all fall in the claimed scope of the invention, and application claims protection domain is defined by its equivalent of appending claims.

Claims

1. contain a screening method for amino small-molecule substance and protein conjugate top condition based on EDC reaction preparation, it is characterized in that: utilize present method that utilization can be found fast to utilize the efficient coupling protein matter molecule of EDC (Chinese: 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate) (containing carboxyl :-COOH freely) with small-molecule substance (containing primary amine group :-NH ₂) optimum reaction condition: the molecule number (ratio of amount of substance) of EDC and protein and small-molecule substance, be a kind of simple and direct, efficient, micro-, be used for screening the scientific approach of optimum experimental condition.

2. following detailed description in detail prepares based on EDC reaction the screening method containing amino small-molecule substance and protein conjugate top condition:

3. the screening method flow process containing amino small-molecule substance and protein conjugate top condition based on EDC reaction preparation according to claim 2, it is characterized in that, step 2) in from No. 1 pipe to No. 11 pipes EDC or micromolecular concentration dilute twice successively.

4. the screening method flow process containing amino small-molecule substance and protein conjugate top condition based on EDC reaction preparation according to claim 2, it is characterized in that, step 2) in, in glove box, take EDC, answer matching while using, and use rapidly after preparing.