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Polyacrylamide gel and application of same in electrophoretic separation of small molecular protein or polypeptide

A polyacrylamide gel and acrylamide technology, applied in the field of biochemistry, can solve the problems of difficulty in popularization, heavy workload, complicated operation, etc., and achieve the effects of easy popularization, reduced workload, and simple operation.

Inactive Publication Date: 2015-11-04
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Glycine-SDS-PAGE is used in both systems, among which the mini-gel is more widely used and easy to operate, but the resolution is lower; the current application of Tricine-SDS-PAGE technology is mostly seen in the large-gel operating system, and the resolution High, but cumbersome to operate
[0005] If you want to separate proteins below 20kDa, you must use Tricine-SDS-PAGE, and at the same time use large plate glue, which will bring two disadvantages: one is cumbersome operation and heavy workload; Glue replacement, Tricine-SDS-PAGE is difficult to promote

Method used

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  • Polyacrylamide gel and application of same in electrophoretic separation of small molecular protein or polypeptide
  • Polyacrylamide gel and application of same in electrophoretic separation of small molecular protein or polypeptide
  • Polyacrylamide gel and application of same in electrophoretic separation of small molecular protein or polypeptide

Examples

Experimental program
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Effect test

Embodiment 1

[0048] Example 1. Preparation of polyacrylamide gel and its application in separating small molecular proteins

[0049] 1) Preparation of polyacrylamide gel

[0050] Prepare polyacrylamide stock solution (AB-3) and electrode buffer solution according to the formulas in Table 1 and Table 2, respectively. The polyacrylamide stock solution is stored at 7-10°C, and the electrode buffer solution is prepared at 20°C. Store at ~25°C (room temperature).

[0051] Table 1 Formula of acrylamide-methylenebisacrylamide stock solution

[0052]

[0053] AB-3 in the table represents an acrylamide-methylenebisacrylamide stock solution with a crosslinking degree of 3%.

[0054] Table 2 Formulas of electrode buffer and gel buffer

[0055]

[0056] In the table, Tris means tris hydroxymethyl aminomethane, Tricine means tris hydroxymethyl glycine, HCl means hydrochloric acid (density 1.20g / mL, mass fraction 36%, 12 mol / L), SDS means sodium dodecylsulfonate.

[0057] Use the formula in Ta...

Embodiment 2

[0077] Embodiment 2, the electrophoresis of small molecular weight protein

[0078] Prepare 4% stacking gel and 18% separating gel according to the ratio in Table 3, and pour the discontinuous gel. Ultra-low molecular weight protein Marker II (Beijing Huitian Oriental Technology Co., Ltd., product number: HT412) (95 ° C, 5min denaturation) and rainbow pre-stained ultra-low molecular weight protein Marker (Beijing Huitian Oriental Technology Co., Ltd., product number: RTD6110) (No denaturation required) Carry out electrophoresis separation after loading the sample, set the voltage according to Table 4, 60V in the stacking gel (4%), 140V in the separation gel (18%), other operations are the same as those in Example 1, and the experimental results are as follows figure 2 shown.

Embodiment 3

[0079] Embodiment 3, the compatibility test of polyacrylamide gel of the present invention and Western Blot

[0080] Lyse 3T3-L1 cell samples (purchased from the American Type Culture Collection, Cat. No.: CL-173), extract protein (total protein) and quantify it with BCA, dilute the protein extract and dilute it with 10 μg (total protein mass) and 15 μg (total protein mass) was loaded for electrophoretic separation. The protein was denatured before loading, that is, the protein 4× loading buffer (produced by Beijing Huitian Oriental Technology Co., Ltd., product number: P1015) was mixed with the extracted protein sample, and placed at 99 ° C for 10 min. After electrophoresis, transmembrane transfer and primary antibody hybridization (the primary antibody of the housekeeping gene β-Actin was purchased from Abmart Company, catalog number: P30002; the primary antibody of the target gene was purchased from Santa Cruz Biotechnology Company, catalog number: sc-30223), two Anti-hybr...

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Abstract

The invention discloses a polyacrylamide gel and application of the same in electrophoretic separation of small molecular protein or polypeptide. The polyacrylamide gel comprises independently prepared spacer gel and separation gel. Each part of the spacer gel comprises the following raw materials: 2.67 mL of water, 0.33 mL of an acrylamide-methylene bisacrylamide solution, 1 mL of a gel buffer, 30 [mu]L of an aqueous ammonium persulfate solution and 3 [mu]L of tetramethyl ethylenediamine. Each part of the separation gel comprises raw materials described in 1) or 2), wherein the raw materials described in 1) are 2.20 mL of water, 1.2 mL of the acrylamide-methylene bisacrylamide solution, 2 mL of the gel buffer, 0.6 mL of glycerol, 30 [mu]L of the aqueous ammonium persulfate solution and 3 [mu]L of tetramethyl ethylenediamine; and raw materials described in 2) are 0.88 mL of water, 2.23 mL of the acrylamide-methylene bisacrylamide solution, 2.23 mL of the gel buffer, 0.66 mL of glycerol, 22 [mu]L of the aqueous ammonium persulfate solution and 2.2 [mu]L of tetramethyl ethylenediamine. An electrode buffer used in the invention is a tris(hydroxymethyl)aminomethane-tris(hydroxymethyl)glycine buffer system. The polyacrylamide gel has the advantages of easy popularization, simple operation, high resolution and good compatibility with immunoblotting and the like.

Description

technical field [0001] The invention relates to a polyacrylamide gel and its application in electrophoresis separation of small molecular proteins or polypeptides, belonging to the technical field of biochemistry. Background technique [0002] Electrophoresis technology is an essential experimental method for the separation and identification of proteins and nucleic acids in the field of biochemistry. The electrophoresis technologies used by almost any scientific research unit include polyacrylamide gel electrophoresis (SDS-PAGE), agarose gel electrophoresis, etc. The former is often used to separate proteins, and the latter is often used to separate nucleic acids. [0003] SDS-PAGE can be divided into Glycine-SDS-PAGE and Tricine-SDS-PAGE due to different buffer systems. Methylaminomethane (Tris)-Trisine (Tricine) Buffer System". The resolution of Glycine-SDS-PAGE is 20-200kD, and the resolution of proteins below 20kD is low, and the bands of small molecules are diffuse, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68
CPCG01N33/68
Inventor 赵春江姜顺严张博吴常信
Owner CHINA AGRI UNIV