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Electrophoretic analysis method for modified protein isolation and identification

A technology for electrophoresis analysis and protein separation, which is applied in the field of separation of modified proteins and natural proteins by electrophoresis, which can solve the problems of complicated operation and high requirements for equipment and equipment, and achieve the effects of simple steps, convenient operation and obvious effect.

Inactive Publication Date: 2012-05-09
NANCHANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, 2D-PAGE is a two-dimensional electrophoresis, which requires high equipment and complicated operation

Method used

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  • Electrophoretic analysis method for modified protein isolation and identification
  • Electrophoretic analysis method for modified protein isolation and identification
  • Electrophoretic analysis method for modified protein isolation and identification

Examples

Experimental program
Comparison scheme
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Embodiment 1

[0018] Separation of iron-chelated ovotransferrin (Holo-OVT) and iron-free ovotransferrin (Apo-OVT).

[0019] Use Urea-PAGE protein electrophoresis to separate iron ion-modified ovotransferrin (Holo-OVT) and iron-free ovotransferrin (Apo-OVT). Their molecular weight is about 76KDa. After ovotransferrin is modified by iron ions, the Iron ions are positively charged and neutralize part of the negative charges in the protein, so iron ion-modified ovotransferrin (Holo-OVT) is less negatively charged than iron-free ovotransferrin (Apo-OVT).

[0020] The specific implementation process includes the following steps.

[0021] 1. Prepare the gel and buffer.

[0022] The gel solution contains 7M Urea, 6% acrylamide (Acr:Bis = 37.5:1), 20mM EDTA, 90mM Tris-boric acid (pH8.4), AP, ddH2O, TEMED.

[0023] After mixing equal volumes of protein solution and loading buffer, the sample contains 50% sucrose, 7M Urea, 20mM EDTA and 90mM Tris-boric acid (pH8.4).

[0024] The electrode buffer co...

Embodiment 2

[0030] Quantitative analysis of the degree of ovotransferrin (OVT) modification.

[0031] Ovotransferrin containing different concentrations of iron ions was used as the protein sample to be analyzed, and iron-free ovotransferrin (OVT) and iron ion-modified ovotransferrin (Fe 2 -OVT) separation. Analysis of Fe by Quality One software 2 -The ratio of OVT to the total ovotransferrin, that is, to quantitatively analyze the degree of modification of ovotransferrin.

[0032] The specific implementation process includes the following steps.

[0033] 1. Prepare the gel and buffer.

[0034] The gel solution contains 6.5M Urea, 5% acrylamide (Acr:Bis = 32.5:1), 20mM EDTA, 90mM Tris-boric acid (pH8.4) and AP, ddH 2 O. TEMED.

[0035] After mixing equal volumes of protein solution and loading buffer, the sample contains 40% sucrose, 6.5M Urea, 20mM EDTA and 90mM Tris-boric acid (pH8.4).

[0036] The electrode buffer contains 25mM EDTA, 112.5mM Tris-boric acid (pH8.4).

[0037]2. S...

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Abstract

The invention discloses an electrophoretic analysis method for modified protein isolation and identification. The method is characterized by being implemented by the following steps of: (1) preparing gel and a buffer solution, (a) a gel solution consists of 6-8M of urea, 4-8 percent of acrylamide, 20mM of EDTA (Ethylene Diamine Tetraacetic Acid), 90mM of Tris (Trihydroxymethyl aminomethane)-boric acid of which the pH is 8.4, AP (Ammonium Persulfate), distilled water and TEMED (Tetramethylethylenediamine), wherein in the acrylamide, the ratio of Acr to Bis is (30-40:1; (b) a loading buffer solution consists of 35-50 percent of cane sugar, 6-8M of urea, 20mM of EDTA and 90mM of Tris-boric acid of which the pH is 8.4; and (c) an electrode buffer solution consists of 25mE of EDTA and 112.5mM of Tris-boric acid of which the pH is 8.4; (2) conventionally cooling under the condition that the electrophoresis voltage is 100-180V and the length of a gel plate is 50-200 millimeters for 4-6 hours till the temperature is below 60 DEG C; and (3) dyeing the gel, decolorizing, imaging, judging a strip of modified proteins in reference to unmodified proteins, or quantifying the modifying degree of proteins by using QualityOne software. The method has simple steps and a remarkable ionophortic separation effect, and is convenient to operate; and the modifying degree of natural proteins can be analyzed.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for separating modified proteins and natural proteins by electrophoresis. Background technique [0002] Protein post-translational modification is an important aspect in protein identification work. Post-translational modifications include phosphorylation, glycosylation, metal chelation, N-terminal blocking, disulfide bond formation, and the like. Protein primary structure can be deduced from gene sequence, but post-translational modification information can hardly be obtained from gene sequence alone. It is well known that these modifications are very important for the function of proteins and even the functions of cells, and are often related to the occurrence of diseases. Therefore, the separation and identification of proteins with different modifications is of great significance in the study of proteome, especially functional proteome. [0003] The most effective met...

Claims

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Application Information

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IPC IPC(8): G01N27/447
Inventor 陈红兵简珊吴志华佟平高金燕李欣杨安树
Owner NANCHANG UNIV