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Preparation method and application of sephadex gel chromatographic column suitable for low-temperature centrifugation

A dextran gel and chromatographic column technology, which is applied in the chemical field, can solve the problems of high commodity prices, long time, and affecting enzyme protein activity, and achieve the effects of shortening desalting time, convenient production, and cost saving

Pending Publication Date: 2019-03-19
JIANGXI AGRICULTURAL UNIVERSITY
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  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The traditional desalination methods are chromatography or dialysis, which are carried out by using commercial dextran chromatography columns or cellulose dialysis bags respectively. The layer (dialysis) analysis takes a long time, the procedure is complicated, the efficiency is low, and the commodity price is relatively expensive
Disadvantages of cellulose dialysis membrane (bag): dialysis must be carried out in a container, which takes a long time (overnight), and the dialysate needs to be replaced several times in the middle. The more samples to be dialyzed, the greater the requirement for the volume of the container. It is a special product and must be purchased from the relevant company
The column is long, usually the column height is above 15cm, and more dextran is needed; the desalination through the filtration of the chromatographic column takes a long time; it cannot be carried out at low temperature (4°C), which will affect the activity of the enzyme protein; the column and Filtration time is long, not suitable for the processing of large batches of samples

Method used

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  • Preparation method and application of sephadex gel chromatographic column suitable for low-temperature centrifugation
  • Preparation method and application of sephadex gel chromatographic column suitable for low-temperature centrifugation
  • Preparation method and application of sephadex gel chromatographic column suitable for low-temperature centrifugation

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preparation example Construction

[0015] 1. Preparation of Sephadex Chromatography Column

[0016] 1.1 Gel pretreatment Weigh about 3g of dextran (such as Sephadex G25), add 50ml of column equilibrium solution, and fully swell at room temperature.

[0017] 1.2 Column packing Take 1 medical injection needle (specification 3ml), put glass wool (grass wool, replace the porous plate of commercial chromatography column) at the bottom to the scale of 0.5ml, and compact it. Inject the swollen Sephadex G25 to the mark of 2.5ml, prevent the generation of air bubbles during the injection process, let it stand for 5 minutes, then place it in a centrifuge at 4°C at 500 rpm for 1min, and set it aside.

[0018] 2. Centrifugal desalting of chromatography column

[0019] Place the prepared chromatographic column in a 10ml or 15ml polyethylene (PE) centrifuge tube, inject the crude enzyme extract to be desalted on the chromatographic column, and centrifuge at 500 rpm at 4°C for 5 minutes. After centrifugation, the The liquid...

Embodiment

[0021] The activities of 3 carbon metabolism-related enzymes (cell wall acid invertase CWIN, sucrose phosphate synthase SPS, sucrose synthase SS) in 4 different parts of tissues (leaf, seed embryo, and seed coat) of 3 cotton lines were detected. By using Compared with the control (not desalted), the dextran chromatography column and the corresponding desalting technology developed by the present invention have significantly improved activities of the four enzymes. The specific results are shown in Table 1, Table 2, and Table 3.

[0022] Table 1 Effect of low-temperature centrifugation and desalting of dextran column on the activity of acid invertase in the cell wall of different plant tissues

[0023]

[0024] Table 2 Effect of low temperature centrifugation and desalting of dextran column on the activity of sucrose phosphate synthase in different plant tissues

[0025]

[0026] Table 3 Effect of low temperature centrifugation and desalting of dextran column on the acti...

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Abstract

The invention discloses a preparation method and application of a sephadex gel chromatographic column suitable for low-temperature centrifugation. The preparation method includes: weighing 3 g of sephadex G-25, adding into 50 ml of a column balance liquid, and allowing full swelling at room temperature; collecting little glass wool with a disposable PE (polyethylene) glove, filling the bottom of asmall medical injection needle tube, compacting until the height reaches 0.5 ml graduation mark of the small medical injection needle tube so that a porous plate of a commercial chromatographic column is replaced, injecting fully swollen sephadex to reach the 2.5 ml graduation mark of the small medical injection needle tube while bubbles are prevented, allowing standing for 5 min, centrifuging for 1 min at 500 r / m at 4 DEG C, and getting ready for use. Compared with traditional desalting appliances and methods, the sephadex gel chromatographic column can provide ideal desalting, allows desalting time to be greatly shortened and experimental cost to be reduced, and is highly worthy of application.

Description

technical field [0001] The invention belongs to the technical field of chemistry, and in particular relates to a preparation method and application of a dextran gel chromatography column suitable for low-temperature centrifugation. Background technique [0002] In plant physiological and biochemical experiments, it is often necessary to detect enzyme activity. The crude enzyme solution extracted from plant materials usually contains many impurities, and too much inorganic salt will seriously inhibit the expression of the catalytic activity of the enzyme protein, and even the activity of the enzyme cannot be detected. The traditional desalination methods are chromatography or dialysis, which are carried out by using commercial dextran chromatography columns or cellulose dialysis bags respectively. The layer (dialysis) analysis takes a long time, the procedure is complicated, the efficiency is low, and the commodity price is relatively expensive. Disadvantages of cellulose di...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): B01D15/22B01D15/10B01D15/26
CPCB01D15/10B01D15/22B01D15/26
Inventor 汤飞宇罗海华
Owner JIANGXI AGRICULTURAL UNIVERSITY
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