Method for extracting and separating whole protein of rat hippocampus, suitable for dimensional electrophoresis

A two-dimensional electrophoresis and whole protein technology, applied in the field of protein separation and identification, can solve the problems of high experimental cost, time-consuming and laborious, etc., achieve good repeatability, good focusing effect, and reduce protein degradation.

Inactive Publication Date: 2014-06-25
HUAZHONG AGRI UNIV
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

The vast majority of samples need to explore the most suitable conditions through multiple

Method used

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  • Method for extracting and separating whole protein of rat hippocampus, suitable for dimensional electrophoresis
  • Method for extracting and separating whole protein of rat hippocampus, suitable for dimensional electrophoresis
  • Method for extracting and separating whole protein of rat hippocampus, suitable for dimensional electrophoresis

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Embodiment Construction

[0042] The materials used in the present invention are: IPG prefabricated strips (Bio-Rad, USA), pH4-7, 17cm, stored in -20°C refrigerator; carrier ampholyte (Bio-Rad, USA), stored in -4°C refrigerator; isoelectric focusing Loading buffer (Bio-Rad, USA), stored in refrigerator at 4°C; Urea, CHAPS, DTT (Dithiothreitol), Bromophenol Blue (Bromophenol Blue); Mineral Oil (Mineral Oil), SDS, Tris, Iodoacetamide; Low melting point agar Sugar, Glycine, Acrylamide; Bisacrylamide (Bis), Ammonium Persulfate; TEMED, Glycerin, Thiourea, Bio-Safe TM Coomassie.

[0043] Instruments and software used in the present invention include: Y96000 type electronic balance (Germany Sartorius); T18 endotype tissue homogenizer (Germany IKA company); ultra-low temperature refrigerator (Thermo fisher); UV-2100 type spectrophotometer (especially Nika (Shanghai) Instrument Co., Ltd.); PROTEIN IEF System (BIO-Rad, USA); PROTHIN IEF Cell (BIO-Rad, USA); horizontal shaker (Changzhou Guohua Electric Co., Ltd.)...

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Abstract

The invention discloses a method which is used for extracting and separating whole protein of rat hippocampus and is suitable for dimensional electrophoresis. The method comprises the following steps: (1) placing a rat in frozen artificial cerebrospinal fluid; (2) pouring liquid nitrogen in a mortar for precooling before grinding; (3) standing turbid liquid at room temperature; (4) subpackaging liquid supernatant; (5) adding RB ( rehydration buffer) into protein conglomeration; (6) making a standard curve by 5, 10, 15, 20, 25, 30 and 35 micrograms of BSA (Bull Serum Albumin); (7) loading analysis gel, namely silver staining, with the quantity shown in the specification; (8) taking out a dried gel strip and rewarming at room temperature; (9) adding coverage oil into an isoelectric focusing disk; (10) performing isoelectric focusing treatment; (11) rewarming the treated gel strip; (12) preparing low melting agarose from upper tank liquid; and (13) carrying out coomassie blue staining on the gel and drawing. The method is easy to implement and is suitable for extracting of rat hippocampus tissue protein samples, the operation is simple and convenient, and a dimensional electrophoretogram with more protein spots, clear horizontal stripes and good repeatability is obtained.

Description

technical field [0001] The present invention relates to the technical field of protein separation and identification, and more specifically relates to a method for extracting and separating whole proteins of rat hippocampal tissue samples, which can effectively separate hippocampal tissue proteins, display protein differences and subsequently identify these proteins, and is suitable for rat nervous system Functionally relevant toxicology studies and drug screening. Background technique [0002] Protein is the main executor of life activities in organisms. With the deciphering of the whole genome sequence of a large number of organisms such as humans and animals and the development of functional genome research, life scientists are paying more and more attention to the composition and activity of proteins in organisms. At present, proteome research is progressing very rapidly in the world, and both basic theory and technical methods are constantly improving and improving. S...

Claims

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Application Information

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IPC IPC(8): G01N1/40G01N1/34
Inventor 王江华李广宇袁勇超党垚李静
Owner HUAZHONG AGRI UNIV
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