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Extracting method for plant chloroplast total protein and special extract therefor

A chloroplast and total protein technology, which is applied in the field of plant chloroplast total protein extraction, can solve the problems of small number of protein spots, horizontal and vertical tailing of two-dimensional electrophoresis, and insufficient extraction of chloroplast total protein.

Inactive Publication Date: 2009-07-08
INST OF BOTANY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The steps of the above conventional protein extraction methods are relatively simple, and the removal of some impurities is not ideal, and there are relatively serious tailings in the horizontal and vertical directions of the two-dimensional electrophoresis graph; in addition, these methods are not comprehensive enough for the extraction of the total chloroplast protein. The number of protein spots is relatively small
This shows that conventional protein extraction methods have limitations in extracting total chloroplast proteins

Method used

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  • Extracting method for plant chloroplast total protein and special extract therefor
  • Extracting method for plant chloroplast total protein and special extract therefor
  • Extracting method for plant chloroplast total protein and special extract therefor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1, Extraction of Salicornia chloroplast total protein and its two-dimensional electrophoresis analysis to chloroplast total protein

[0027] 1. Cultivation of Salicornia

[0028]Sow the seeds of Salicornia (S.europaea Linn.) (Jiangsu Jinglong Marine Industry Development Co., Ltd.) on the vermiculite soaked in water, put them in the greenhouse, and pour the Hoagland nutrient solution once a week after it germinates. The solution formula refers to the method of Hoagland et al. (Hoagland, DR., Arnon DI.Calif Agric Exp Stn Circ.1950, 347, 1-39), 200mM NaCl is added to the nutrient solution after sowing 30 days to maintain the salt Horngrass normal growth state. Fifty days after sowing, Salicornia was collected for chloroplast extraction. Growth conditions: the daytime temperature is set at 25°C, the nighttime temperature is set at 20°C, the relative humidity is 50±10%, and the daily light time is 16 hours.

[0029] 2. Extraction of Salicornia chloroplast and iden...

Embodiment 2

[0047] Example 2, Extraction of Arabidopsis thaliana total chloroplast protein and its two-dimensional electrophoresis and mass spectrometry analysis of chloroplast total protein

[0048] 1. Culture of Arabidopsis

[0049] Arabidopsis thaliana (A. thaliana) wild type (Columbia ecotype) was grown in an artificial climate box, watered every three days, the growth conditions were 16 hours of light, 8 hours of darkness, the temperature was 23°C, and the relative humidity was 70%. After seven weeks of growth, Arabidopsis leaves were collected for chloroplast extraction.

[0050] 2. Extraction of Arabidopsis chloroplasts

[0051] Extract according to the method for extracting Salicornia chloroplast in Example 1. 8-12g of Arabidopsis leaves are extracted to obtain 0.1-0.3g of Arabidopsis chloroplasts.

[0052] 3. Extraction of Arabidopsis chloroplast total protein

[0053] The specific steps are as follows: 1mL chloroplast total protein extract (the solute in the chloroplast prot...

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Abstract

The invention discloses an extracting method for a chloroplast total protein and a special extracting solution thereof. The special extracting solution for the chloroplast total protein is the aqueous solution which contains the following matters with the end concentration: 90 to 100mM ethylenediaminetetraacetic acid, 90 to 100mM trihydroxymethyl aminomethane, 40 to 60mM sodium tetraborate, 0.9 to 1.1 volume percent Triton X-100, 1.9 to 2.1 volume percent beta-mercaptoethanol, and 28-32g / 100ml sucrose. The high quality chloroplast total protein obtained through the special extracting solution and extracting method solves the problem of preparing the chloroplast total protein sample in the dielectrophoresis, and simultaneously has the mass spectrum compatibility. The method can be applied to the proteomics study as a general chloroplast total protein extracting method.

Description

technical field [0001] The invention relates to a method for extracting plant chloroplast total protein and a special extracting solution thereof. Background technique [0002] As one of the most important organelles with complex membrane structure, chloroplast exists in all higher plants and some algae. From a biochemical point of view, chloroplasts can be subdivided into different subcomponents, each of which contains proteins that perform specific functions. In order to elucidate the types of proteins in different subcomponents of chloroplasts, various methods for isolating chloroplast subcomponents and proteomics techniques have been combined to study the outer capsule, inner capsule, matrix, thylakoid membrane and thylakoid cavity of chloroplasts. The analysis of proteomics provides detailed information on protein profiles in each subfraction. Although the identity and function of proteins in the different subfractions of chloroplasts is well understood, this method o...

Claims

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Application Information

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IPC IPC(8): C07K14/415C07K1/14G01N33/68
Inventor 李银心范鹏祥
Owner INST OF BOTANY CHINESE ACAD OF SCI
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