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Charge-mass double-focusing two-dimensional gel electrophoresis separation method and its kit

A gel electrophoresis and electrophoresis separation technology, applied in the field of protein gel electrophoresis separation, can solve the problems of poor resolution ability of basic proteins, influence of second-dimensional mass separation ability, easy protein precipitation, etc., to overcome protein precipitation and low cost , the effect of improving the resolution

Active Publication Date: 2017-06-13
HUAZHONG NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantages of isoelectric focusing mainly include two aspects: (1) the resolution ability of basic proteins is poor; (2) proteins are easy to precipitate
Moreover, the molecular weight of different isoforms of histones is very similar, which makes the ability of mass separation in the second dimension also affected

Method used

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  • Charge-mass double-focusing two-dimensional gel electrophoresis separation method and its kit
  • Charge-mass double-focusing two-dimensional gel electrophoresis separation method and its kit
  • Charge-mass double-focusing two-dimensional gel electrophoresis separation method and its kit

Examples

Experimental program
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Effect test

Embodiment 1

[0045] The rat liver histones were separated by the first-dimensional charge electrophoresis separation method. The specific operation steps are as follows:

[0046] (1) Extraction of rat liver histones: First, the rat liver tissue was washed with normal saline to remove blood, then homogenized with nuclear separation reagent, and washed with washing solution without Tween X-100 and NP40 after centrifugation. The isolated cell nuclei were extracted with 0.2M sulfuric acid solution, precipitated with trichloroacetic acid, washed with ice acetone after centrifugation, and freeze-dried to obtain histone biological sample freeze-dried powder. The components of the nuclear separation reagent are: Tris-HCl (15mM), KCl (60mM), CaCl 2 (11mM), NaCl (5mM), MgCl 2 (5mM), Sucrose (250mM), DTT (1mM), Sodium Butyrate (10mM), NaN 3 (0.02 wt%), 0.4 wt% NP 40 and 0.80 wt% Tween-X100. Except that the washing solution does not contain Tween and NP40, other components are the same as the nucle...

Embodiment 2

[0053] The rat liver histones were separated by charge-mass double-focusing two-dimensional gel electrophoresis separation method. The specific operation steps are as follows:

[0054] Step (1)~step (3) are with embodiment 1;

[0055] (4) Mass separation electrophoresis: cut off the first-dimensional charge electrophoresis separation gel strip obtained in step (3), place it on the sodium dodecyl sulfonate-polyacrylamide separation gel, and fill the gaps on both sides. Add blank Tween-acetic acid-urea separation gel (TAU gel) to ensure uniform current distribution, add 50 μl indicator, and then add electrophoresis solution to soak the entire separation gel, apply voltage, and start electrophoresis: run at 70 volts for 1 hour, Then change the voltage to 120 volts to continue electrophoresis. When the indicator runs out of the electrophoresis tank, the electrophoresis is stopped to obtain a charge-mass two-dimensional electrophoresis separation gel strip;

[0056] The ratio of e...

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Abstract

The invention belongs to the field of protein gel electrophoresis separation, and particularly relates to a charge-mass double-focusing two-dimensional gel electrophoresis separation method and a kit thereof. According to the electrophoresis separation method, under the action of selective coordination of Cu<2+> and amino acid residues in protein under an acidic condition, not only is the protein positively charged due to protonation of the basic amino acid residues, but also similar histone isomers of an amino acid sequence are positively charged differently due to a selective coordination function of metal ions, after direct current is applied, the histone isomers with different positive charges have different migration rates, so that separation is achieved; and after one-dimensional charge separation, SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) can be further performed, and the charge-mass double-focusing two-dimensional gel electrophoresis is used for separation of the histone isomers. The electrophoresis separation method is simple in operation process, high in resolution and low in cost, and defects of high probability of protein precipitation and analysis limitation of the histone isomers during isoelectric focusing electrophoresis in the prior art are overcome.

Description

technical field [0001] The invention belongs to the field of protein gel electrophoresis separation, in particular to a charge-mass double-focusing two-dimensional gel electrophoresis separation method and a kit thereof. Background technique [0002] Histones are basic proteins in eukaryotic chromatin, and also the material basis of epigenetics. A large number of studies have shown that histones are closely related to the occurrence and development of many major human diseases. Therefore, the isolation and identification of histones is important for understanding basic physiology and pathology. process is important. However, the structural similarity of various histone isoforms and the presence of numerous post-translational modifications make analytical techniques challenging. [0003] In the existing gel electrophoresis technology, isoelectric focusing is generally used for first-dimensional separation, and then sodium dodecyl sulfonate-polyacrylamide gel electrophoresis ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/435C07K1/26
CPCC07K14/47
Inventor 钟鸿英张文洋唐雪妹郑石黄璐璐
Owner HUAZHONG NORMAL UNIV
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