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Non-denaturing preparative electrophoresis

An electrophoresis device and electrophoresis separation technology, applied in the field of biochemistry, can solve the problems of limiting the application of electrophoresis technology, high cost, unsatisfactory and other problems

Inactive Publication Date: 2008-12-24
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0012] Although there are electrophoresis methods that can be used for the preparation of a small amount of protein or samples, unfortunately, so far, when electrophoretic separation technology is applied to the production and preparation of small and large-scale protein samples in the laboratory, no matter in terms of separation accuracy or other The degree of practicality is far from satisfactory (failed to reach the level of protein analysis), because these electrophoresis methods are difficult to achieve batch continuous production, the devices and methods are relatively complicated, and the cost is high, which greatly limits the Application of this electrophoresis technique in protein separation and purification

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Example 1 Investigation of electrophoretic behavior after adding pigments to proteins with different molecular sizes

[0061] 1 Experiment design purpose: The non-denaturing electrophoresis of the present invention indicates the electrophoretic behavior of the target protein by adding a sample buffer containing Coomassie brilliant blue dye to the sample, that is, plate B shows the electrophoretic position of the target protein, and plate A collects the purpose according to the instructions of plate B protein, but does the loading buffer containing Coomassie Brilliant Blue dye change the electrophoretic migration behavior of the sample protein? Is the electrophoretic migration of the protein of interest altered in the mounting buffer with and without the Coomassie Brilliant Blue dye? The experiment investigated protein molecules of different sizes, thymosin 28 peptide (3.2kd), recombinant human serum albumin thymosin fusion protein (69kd), recombinant asparaginase (144kd...

Embodiment 2

[0086] Example 2: Separation of Pichia pastoris fermented recombinant human serum albumin thymosin fusion protein sample by non-denaturing preparative electrophoresis Purification and activity determination of non-denaturing electrophoresis

[0087] 1 Sample pretreatment:

[0088] Same as Example 1

[0089] 2 Electrophoresis conditions

[0090] 1 electrophoresis buffer

[0091] Same as Example 1

[0092] 2 Non-denaturing loading buffer:

[0093] Same as Example 1

[0094] 3 Electrophoresis operation process, refer to literature [7] Carry out, that is: add the same amount of sample to A and B loading buffer respectively, but without boiling, directly centrifuge at 8000r / min for 10min and then load the sample for electrophoresis, in which the electrode current: constant current of stacking gel 15mA, constant current of separating gel 30mA, in When the indicated target fusion protein is approaching the bottom of the collection tank, collect samples at intervals of 15 minute...

Embodiment 3

[0102] Example 3: Using non-denaturing preparative electrophoresis to separate Escherichia coli fermented recombinant asparaginase samples, non-denaturing electrophoresis purification and activity determination

[0103] 1 Sample pretreatment:

[0104] Same as Example 1

[0105] 2 Electrophoresis conditions

[0106] 1 electrophoresis buffer

[0107] Same as Example 1

[0108] 2 Non-denaturing loading buffer:

[0109] Same as Example 1

[0110] 3 Electrophoresis operation process, refer to the literature for the operation process [7] Carry out, that is: add the same amount of sample to A and B loading buffer respectively, but without boiling, directly centrifuge at 8000r / min for 10min and then load the sample for electrophoresis, in which the electrode current: constant current of stacking gel 15mA, constant current of separating gel 30mA, in When the indicated target fusion protein is approaching the bottom of the collection tank, collect samples at intervals of 15 minute...

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Abstract

The invention relates to an electrophoretic separation device and a technology used for laboratorial and large-scale separated and purified biological samples. The invention mainly relates to the electrophoretic separation device which is a device capable of being used for performing the non-denaturing electrophoretic preparation technology, and a method required for completing the electrophoretic preparation technology by the device. The device mainly comprises a cooling chamber which is provided with a good heat exchange structure, a vertical electrophoretic plate with non-denaturing polyacrylamide gel arranged in the cooling chamber, a concentrating and collecting chamber which is used for sample collection, and an electrophoretic cell. The technology mainly comprises screening of collection time of purified proteins and a method for collecting the purified proteins. The device and the technology have the characteristics of large processing capacity, high resolution, convenient operation and so on, can be used for preparing laboratorial and large-scale biological products and biological medicines, and have considerable economic benefit and social benefit.

Description

technical field [0001] The invention relates to a novel electrophoresis separation device for separating proteins and its technology, belonging to the technical field of biochemistry. technical background [0002] In 1808, the phenomenon of electrophoresis (EI electrophoresis) had already been discovered, but it was not until 1937 that Tiselius of Sweden established the "free electrophoresis method" and successfully divided serum proteins into five main components, which made electrophoresis technology widely used in biochemical analysis. Applications. Since then, in 1984, Weiland et al. developed an electrophoresis method using paper as a support, which made the electrophoresis technology further applied and developed. After continuous efforts, especially in the past 30 years of development, electrophoresis technology has been greatly improved from theory to practice, and has become an indispensable method in protein and nucleic acid analysis. [1] . [0003] There are ma...

Claims

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Application Information

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IPC IPC(8): C07K1/26B01D57/02G01N27/447
Inventor 陈建华张新国闫璐颖唐莉
Owner CHINA PHARM UNIV
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