Method for concentrating and desalting urine sample

A urine and sample technology, applied in the field of biological detection, can solve the problems of inability to effectively concentrate the original sample, poor desalination effect, protein degradation, etc., and achieve the effects of reduced salt concentration, increased process, and more protein loss

Inactive Publication Date: 2011-07-06
李顺民 +1
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Problems solved by technology

Moreover, the voltage often needs to reach more than 5000V during isoelectric focusing. If the salt content is too high, the current will increase during focusing, often exceeding 50μA, which will not only damage the rubber strip but also cause the risk of being broken down by the current.
[0006] There are mainly two types of existing "urine desalination methods": the first method is to use dialysis bags to desalt first, and then extract with organic solvents (including acetone, ethanol, trichloroa

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  • Method for concentrating and desalting urine sample
  • Method for concentrating and desalting urine sample
  • Method for concentrating and desalting urine sample

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[0029] The present invention will be further described below in conjunction with specific examples, but it should not be construed as a limitation of the present invention.

[0030] Take the processing of the urine sample of IgA nephropathy patient as example to illustrate the present invention:

[0031] 1. Collect 20ml of mid-section urine from the patient and freeze it at -80°C for later use;

[0032] 2. Add 3uL 200mM phenylmethylsulfonyl fluoride (PMSF) to every 10ml of urine to prevent proteolysis;

[0033] 3. Divide the above 20ml mixed urine into two tubes and centrifuge at 15000g for 5-10 minutes at 2°C-4°C;

[0034] 4. Collect the supernatant and filter it through a 0.45u nylon filter to remove cell nuclei and bacteria;

[0035] 5. The filtered urine is further concentrated and desalinated by cold acetone or trichloroacetic acid (TCA), centrifuged at 3000g for 10-30 minutes at 2°C-4°C, and the precipitate is taken and dried in the air for 5-10 minutes ;

[0036] 6....

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Abstract

The invention relates to a method for concentrating and desalting a urine sample prior to two-dimensional polyacrylamide gel electrophoresis, belonging to the technical field of biological detection. Certain procedures are improved on the basis of the conventional method, and the method for concentrating and desalting the urine sample comprises the following steps of: firstly, performing ultracentrifugation and organic solvent extraction; then, performing dialysis bag desalting and organic solvent extraction. In this way, the salinity is obviously reduced in comparison with the salinity obtained by using the original treatment method. Moreover, the focusing quantity in subsequent isoelectric focusing can reach 60000 vhr, which is beneficial to the successful completion of isoelectric focusing, and the picture forming quality of SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis) is improved.

Description

technical field [0001] The patent application of the present invention relates to a method for concentrating and desalting urine samples before two-dimensional polyacrylamide gel electrophoresis, which belongs to the technical field of biological detection. Background technique [0002] Gel electrophoresis, based on the migration of charged proteins in an electric field, is a method commonly used in the biomedical field to separate, identify and purify proteins. The advantage is that proteins are isolated and purified simultaneously, allowing researchers to quickly identify the amount of different proteins in a mixture or the purity of a particular protein preparation. Furthermore, key properties of a protein such as isoelectric point and approximate molecular mass can be determined. Among them, polyacrylamide gel electrophoresis (referred to as PAGE) has a high separation ability and is widely used in the separation and identification of proteins. As a molecular sieve, po...

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Application Information

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IPC IPC(8): G01N1/40G01N1/34G01N27/447
Inventor 李顺民祁爱蓉
Owner 李顺民
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