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Multi-dimensional series gel electrophoresis system, electrophoresing method and kit

A gel electrophoresis, multi-dimensional technology, applied in the field of protein gel electrophoresis separation, can solve the problems of reduced acrylamide polymerization reaction ability, single gel properties, unsuitable for basic protein separation, etc., and achieves easy quality control and industrialization, Overcome the effects of single gel structure and easy control of electrophoresis voltage

Active Publication Date: 2017-04-26
HUAZHONG NORMAL UNIV
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  • Abstract
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Problems solved by technology

The disadvantages of conventional isoelectric focusing and polyacrylamide gel electrophoresis are mainly manifested in six aspects: (1) isoelectric focusing is not suitable for basic or acidic proteins, and expensive gel strips and gels are required for isoelectric focusing. Special equipment; (2) The existing polyacrylamide gel has a single structure, only acrylamide and N, N'methylenebisacrylamide monomers, so the prepared gel has a single property, and only depends on the mesh size and (3) It is not suitable for the separation of basic proteins. The existing catalysts ammonium persulfate APS and TEMED can only initiate polymerization under alkaline conditions, while basic proteins Solubility decreases under alkaline conditions, prone to precipitation
Basic proteins are usually dissolved in acidic solutions, but APS and TEMED are protonated under acidic conditions, which reduces the ability to initiate acrylamide polymerization, resulting in incomplete polymerization, long polymerization time, and poor elasticity of the resulting gel; (4) Poor separation ability for proteins with similar structure and molecular weight, especially not suitable for the separation of histones; (5) Poor separation ability for protein isoforms with different post-translational modifications
Taking basic histone as an example, due to the existence of a large number of post-translational modifications, the molecular weights of its different isomers are very similar, which makes conventional polyacrylamide gel electrophoresis can only separate a limited number of protein bands; (6) conventional The fixative and destaining solutions used after polyacrylamide gel electrophoresis are not suitable for basic proteins, because the acidic fixatives and destaining solutions will cause the loss of basic proteins

Method used

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  • Multi-dimensional series gel electrophoresis system, electrophoresing method and kit
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  • Multi-dimensional series gel electrophoresis system, electrophoresing method and kit

Examples

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preparation example Construction

[0056] (1) Chelated Zr 4+ Preparation of a novel monomer: adenosine triphosphate disodium salt (ATPNa 2 ) 1g, allyl glycidyl ether (AGE) 190μL was added to 20mL distilled water, stirred at 40°C for 3 hours to obtain Zr 4+ the monomer;

[0057] (2) Preparation of gel preparation solution: 1.8 g of urea, 1.25 mL of 60% acrylamide / 0.4% N,N'-methylenebisacrylamide, 250 μL of glacial acetic acid, 1.86 mL of distilled water and 10% Triton X-100 solution After 186 μL was mixed well, add ZrCl 4 Solution 10μL, vortex to mix evenly, then add the chelated Zr obtained in step (1) 4+ 100 μL of the new monomer, after mixing evenly, add 140 μL of 10wt% ammonium persulfate and 30 μL of tetramethyldiethylamine in sequence;

[0058] (3) Immobilized Zr 4+ Preparation of polyacrylamide gel: pour the gel preparation solution prepared in step (2) into the gel tank, on the N-hydroxyethylacrylamide-acrylamide copolymer gel, so that the height of the gel tank is the total 1 / 6 of the height, to a...

Embodiment 1

[0063] For the electrophoretic separation and mass spectrometric identification of casein and dephosphorylated casein, multidimensional tandem gel electrophoresis is first performed, and the steps are as follows:

[0064] (1) Sample preparation of casein and dephosphorylated casein: Dissolve the standard sample in the sample buffer solution, which is acidic and can protonate the basic amino acid residues in the sample, so that the sample is positive Charge; the composition of the sample buffer solution is: urea (0.36g), glacial acetic acid (50 μL), 0.2% Pyronin Y (60 μL) and 490 μL of purified water, making its concentration approximately 5 μg / μL;

[0065] (2) Mix the sample solution obtained in step 1) with the auxiliary sample buffer solution at a ratio of 1:1, and the composition of the auxiliary sample buffer solution is CuSO 4 (4μg / μL);

[0066] (3) Add the electrophoresis solution (aqueous acetic acid solution with a volume concentration of 5%) to the multidimensional t...

Embodiment 2

[0071] Separation and mass spectrometry identification of mouse brain histones. The complex protein mixture is first separated according to different properties on the multidimensional tandem gel electrophoresis system of the present invention, and then the protein bands separated by electrophoresis are identified by mass spectrometry. The specific operation steps are as follows :

[0072] (1) Repeat the operation steps (1) to (5) of Example 1, take out the gel obtained in step (5), scan it, and the image is as follows Figure 4 shown;

[0073] (2) Take out the gel obtained by multidimensional tandem gel electrophoresis separation, and cut out relevant swimming lanes;

[0074] (3) The protein strips obtained in step (2) were subjected to in-gel trypsin hydrolysis with trypsin, and the extracted peptides were analyzed by MALDI-MS, and the obtained mass spectrum was as follows Figure 5 shown.

[0075] Figure 4 and Figure 5 The results show that: the multidimensional tand...

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Abstract

The invention belongs to the field of protein gel electrophoresis separation, and particularly relates to a multi-dimensional series gel electrophoresis system, an electrophoresing method and a kit. The multi-dimensional series gel electrophoresis system comprises a first-dimensional TiO2-polyacrylamide compound gel electrophoresis, a second-dimensional N-hydroxyethyl acrylamide-acrylamide copolymerized gel electrophoresis and a third-dimensional immobilized Zr<4+> polyacrylamide gel electrophoresis, wherein the electrophoresis gel is prepared by sequentially filling an electrophoresis tank with the TiO2-polyacrylamide compound gel, the N-hydroxyethyl acrylamide-acrylamide copolymerized gel, the immobilized Zr<4+> polyacrylamide gel and sample spacer gel. The multi-dimensional series gel electrophoresis system is applicable to separation of basic protein mixture with good separation effect and stable quality, is compatible with downstream mass spectrum identification with relatively small interference, is suitable for studies on large-scale proteome, especially separation of basic histone isomers, and is convenient for quality control and industrialization.

Description

technical field [0001] The invention belongs to the field of protein gel electrophoresis separation, and in particular relates to a multidimensional tandem gel electrophoresis system, an electrophoresis method and a kit. Background technique [0002] Polyacrylamide gel is a classic support medium for electrophoretic separation of proteins. Before the polymerization reaction, by adjusting the concentration ratio of acrylamide and N, N'methylenebisacrylamide monomers, polyacrylamide gels with different degrees of cross-linking structure, mesh size and Shaped high molecular polymers, after applying DC voltage, proteins with different molecular weights and charges have different swimming speeds and thus can be separated on polyacrylamide gels. [0003] In the existing gel electrophoresis technology, the first-dimensional separation is carried out according to the charge through the isoelectric focusing technology, and then the second-dimensional separation is carried out accordi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K1/28
CPCC07K1/285
Inventor 钟鸿英张文洋游萍萍庞晨梦
Owner HUAZHONG NORMAL UNIV
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