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Integral immobilization pH gradient production method and application thereof

An integral, gradient technology, applied in peptide preparation methods, chemical instruments and methods, organic chemistry, etc., to achieve stable gradient, low cost, and easy operation.

Inactive Publication Date: 2008-10-29
杨春
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Aiming at the shortcomings of the existing M-IPG, the present invention provides a preparation method that can realize a high-resolution pH gradient in an arbitrary setting range of 1 to 14, and the application of this pH gradient in the separation of ampholytes by isoelectric focusing

Method used

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  • Integral immobilization pH gradient production method and application thereof
  • Integral immobilization pH gradient production method and application thereof
  • Integral immobilization pH gradient production method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1: Weigh (quantify) the medicines listed in Table 1 with serial numbers 1-22, put them in 22 1L volumetric flasks, stir them in an ice-water bath to make a solution, and store it in a refrigerator at -20°C. When using a pipette, take out the required amount for use. This series of solutions can establish a pH gradient between pH 2-11. The polymerization reaction is carried out in a 60°C water bath, and the polymerization time is 5 to 48 hours.

Embodiment 2

[0041] Example 2: Compared with Example 1: In this example, the concentrations of acid and basic monomers used are different while keeping the concentrations of neutral monomers and initiators unchanged. By increasing the amount of AMPS in the serial numbers 1, 2, and 3 in Table 1 to reach 20, 18, and 16 grams, respectively, the pH range was extended to pH=1 in the low pH direction; by increasing the serial numbers 20 and 21 in Table 1 The amount of DEAEMA in, 22 is 230, 270, and 300 grams, respectively, so that the pH range is extended to pH=14 in the high pH direction. At the same time, the polymerization temperature and time remain unchanged.

Embodiment 3

[0042] Example 3: Compared with Examples 1 and 2: This example uses different acidic monomers (such as vinyl sulfonic acid or sodium vinyl sulfonate) and basic monomers (such as vinyl amine). At the same time, the polymerization reaction The temperature and time remain unchanged, so as to achieve a gradient between pH=1-14.

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Abstract

The invention discloses a preparing method and application for an integral immobilized pH gradient; the preparing method comprises the following steps: a series of solution which consist of acid, alkali and neutral monomer and have different pH values are prepared, each solution is polymerized so as to obtain a pH gradient, a plurality of gradients are combined to obtain a pH gradient with wide range, the pH gradients are fixed in a column container by adopting an in-situ polymerization method or a method for polymerization firstly and immobilization then, and so the integral immobilized pH gradient is obtained; compared with the prior art, the method of the invention can conveniently prepare the immobilized pH gradient with any range of 1 to 14 without using commercial amphoteric electrolyte, and has simple operation and low cost; the prepared pH gradient is used for separating the amphoteric electrolytes of protein and polypeptide, etc. by isoelectric focusing, has stable gradient and high resolution, can be used repeatedly and is applicable to purifying equipment.

Description

Technical field: [0001] The invention relates to a preparation method of an integral immobilized pH gradient and its application in isoelectric focusing separation of amphoteric electrolytes, belonging to the technical field of amphoteric electrolyte separation media. Background technique: [0002] Modern proteome separation technology is mainly based on two-dimensional polyacrylamide gel electrophoresis (2D PAGE) and strong cation exchange-reversed-phase high performance liquid chromatography (SCX RP-HPLC), the latter of which is also known as shotgun technology. So far, 2DPAGE is still the most powerful proteomic separation technology, but it has the disadvantages of time-consuming and difficult operation. The invention of immobilized pH gradient (IPG) greatly improves the reproducibility of 2D PAGE, and can increase the amount of sample loaded, thereby improving sensitivity. Users can flexibly select or deploy different ranges of gel strips according to the actual situation. N...

Claims

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Application Information

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IPC IPC(8): G01N27/447C07K1/28
Inventor 杨春
Owner 杨春
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