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Method for improving the stability of polyacrylamide gel premix, premix and application thereof

A polyacrylamide gel and acrylamide technology, which is applied in the biological field, can solve the problems that polyacrylamide is prone to hydrolysis, reduces the resolution of protein bands, and the premix has poor stability and cannot be stored for a long time.

Active Publication Date: 2022-03-04
上海碧云天生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The traditional discontinuous system polyacrylamide gel needs to be prepared immediately: first mix the components of the separating gel and then cast, wait for solidification, then mix the components of the concentrated gel before casting, and wait for the solidification before pouring. The preparation is completed, the process is cumbersome and the waiting time is too long, which is not conducive to the rapid progress of the experiment
Although there are premix products for polyacrylamide gel preparation on the market, the components of the separating gel and stacking gel are mixed well in advance, but due to the high pH of the separating gel in the polyacrylamide gel electrophoresis system, it is easy to cause acrylamide Hydrolysis, the poor stability of the premix solution cannot be stored for a long time
Moreover, polyacrylamide is also prone to hydrolysis after long-term storage of the prepared gel, which reduces the resolution of protein bands during sample electrophoresis and causes bands to be blurred.
In patent CN 102292633B, triethanolamine is added to the premix, and in patent CN108627564A, taurine and HEPES are added to the premix, which properly prolongs the storage time, but still limits the validity period of the product, and the cost of raw materials is relatively high
Some premix products are not compatible with the Tris-Glycine electrophoresis solution commonly used in the laboratory, and additional matching electrophoresis buffers need to be purchased, which is not universal

Method used

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  • Method for improving the stability of polyacrylamide gel premix, premix and application thereof
  • Method for improving the stability of polyacrylamide gel premix, premix and application thereof
  • Method for improving the stability of polyacrylamide gel premix, premix and application thereof

Examples

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preparation example Construction

[0067] As a preferred mode, the gel of the present invention is a discontinuous gel, and the preparation method of the gel comprises: preparing a separation gel premix, adding ammonium persulfate and tetramethylethylenediamine and mixing , join in the glue-making device (such as glue-making glass plate / plastic plate etc.), obtain separating gel gel; The separation gel in the gel making device is integrated to obtain the discontinuous gel; preferably, the sample loading hole of the protein is located in the stacking gel.

[0068] As some alternatives, the basic components that make up the gel, such as Acr-Bis, can be prepared first, and then soaked in a mixed solution containing N,N-dimethylglycine and D-glutamic acid; preferably, Acr-Bis -Bis is prepared together with N,N-dimethylglycine and D-glutamic acid, etc. and mixed thoroughly.

[0069] The present invention also includes the polyacrylamide gel prepared by the method, and the obtained polyacrylamide gel is also a new t...

Embodiment 1

[0093] Example 1, 8% Acr-Bis (29:1) premix preparation and gel electrophoresis based on formula A

[0094] In this example, the preparation and gel electrophoresis of 8% Acr-Bis (29:1) premix based on formula A, the main steps are as follows:

[0095] (1) To prepare the separation gel premix, take the following reagents and place them in a 1000ml beaker: 267ml of 30% Acr-Bis (29:1), 20.6g of N,N-dimethylglycine, 5.88g of D-glutamic acid, SDS 0.5g, HEPES 4.77g, Tris powder 12.1g. Add about 600ml of ultrapure water into the beaker, stir and mix well, add 1M HCl to adjust the pH of the solution to 6.5, and set the volume to 1L; in the solution obtained after the above preparation, the content of each component: N,N-dimethyl Glycine 200mM, D-glutamic acid 40mM, 0.05% sodium dodecyl sulfate (SDS), HEPES 20mM, Tris 100mM;

[0096] (2) Take 10ml of the premix prepared in (1), add 0.1ml of 10% ammonium persulfate (ammonium persulfate substitute) solution, 6μl TEMED, mix well and add...

Embodiment 2

[0102] Example 2, preparation and gel electrophoresis of 10% Acr-Bis (29:1) premix solution based on formula A

[0103] In this example, the main steps of preparation and gel electrophoresis based on the 10% Acr-Bis (29:1) premix of formula A are as follows:

[0104] (1) To prepare the separation gel premix, take the following reagents and place them in a 1000ml beaker: 333ml of 30% Acr-Bis (29:1), 20.6g of N,N-dimethylglycine, 5.88g of D-glutamic acid, SDS 0.5g, HEPES 4.77g, Tris powder 12.1g. Add about 600ml of ultrapure water into the beaker, stir and mix well, add 1M HCl to adjust the pH of the solution to 6.5, and set the volume to 1L; in the solution obtained after the above preparation, the content of each component: N,N-dimethyl Glycine 200mM, D-glutamic acid 40mM, 0.05% sodium dodecyl sulfate (SDS), HEPES 20mM, Tris 100mM;

[0105] (2) Take 10ml of the premix prepared in (1), add 0.1ml of 10% ammonium persulfate solution, 5μl of TEMED, mix thoroughly and add to a 1....

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Abstract

The invention provides a method for improving the stability of polyacrylamide gel premix, the premix and application thereof. The present invention provides an optimized polyacrylamide gel premix formula, adding N,N-dimethylglycine and D-glutamic acid to enhance the stability of the premix, and extend its validity period from about 90 days to about 450 days or longer; the polyacrylamide gel prepared with this master mix has the advantages of low cost, easy operation, fast electrophoresis speed, high resolution, and compatibility with general-purpose Tris‑Glycine electrophoresis buffer, etc.

Description

technical field [0001] The invention belongs to the field of biotechnology, and more specifically, the invention relates to a method for improving the stability of a polyacrylamide gel premix, a premix and an application thereof. Background technique [0002] Polyacrylamide Gel Electrophoresis (PAGE for short), originally proposed by Laemmli, U.K. (Nature 227: 680, 1970), is an electrophoresis technique using polyacrylamide gel as a supporting medium. The network structure has a molecular sieve effect, and is often used to separate proteins and small fragments of nucleic acids. It is available in two formats: non-denaturing polyacrylamide gels and denaturing polyacrylamide gels. During the electrophoresis process of non-denaturing polyacrylamide gel, the tertiary structure of the protein remains intact, and the protein is separated according to the molecular weight, shape and charge gradient of the protein. The denatured polyacrylamide gel contains a strong reducing agent ...

Claims

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Application Information

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IPC IPC(8): C07K1/26
CPCG01N27/44747
Inventor 葛新建李扬
Owner 上海碧云天生物技术有限公司
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