Method for coagulating protein in liquid

Abstract

The invention relates to a method for separating out and condensing specific protein in liquid for dyeing and detection. The liquids include but are not limited to human samples, human blood, saliva, urine and the like. The liquid is diluted and its pH is raised or lowered to form a mixed liquid. Placing the mixed solution in an electrostatic field, moving the specific protein in the mixed solution to one side of the mixed solution, accurately dropwise adding an acidic or alkaline solution, adjusting the pH value of the mixed solution, enabling the pH value of the mixed solution to reach the isoelectric point of the specific protein, and enabling the specific protein to be separated out and precipitated on one side of the mixed solution. In addition, according to the sequence of the pH values from large to small or from small to large, the isoelectric points of all the specific proteins can be traversed in the process, and all the specific proteins can be separated out and precipitated in the same area of the mixed solution.

Description

Technical Field

[0001] The invention belongs to the technical field of protein isoelectric point precipitation, and particularly relates to a method for separating and precipitating various proteins in a liquid by utilizing an electrostatic field. Background Art

[0002] Human samples contain a variety of proteins, some of which are present only in diseased states or abnormally increased in number, and are therefore classified as abnormal proteins. Separating and aggregating these abnormal proteins from human samples such as blood for staining and analysis is of particular significance.

[0003] Existing products mainly use immunological methods to identify various abnormal proteins. However, immunological testing has disadvantages such as high cost.

[0004] Various proteins in human samples have different isoelectric points.

[0005] When the pH value of the solution is higher or lower than the isoelectric point of the protein, the protein in the solution is negatively charged or positively charged respectively. When the solution is placed in an electrostatic field, these charged proteins move toward the two poles and finally gather on both sides of the solution.

[0006] Raise or lower the pH of the solution above or below the isoelectric point of all the proteins we are interested in, so that all the proteins we are interested in will move to one side of the solution under the action of the electric field.

[0007] At their isoelectric point, proteins no longer carry any charge and repel each other, resulting in precipitation. [Note 1]

[0008] [Note 1: P Filho, G Hirata, É Watanabe, E Miranda, 2.49 - Precipitation and Crystallization, ScienceDirect, Volume 2, 2019, Pages 725-738.]

[0009] In the production of pulse protein (pea, chickpea, lentil and other legume proteins), isoelectric precipitation is the most commonly used method [Note 2]

[0010]

Note 2: Karaca, AC, Low, N., & Nickerson, M. (2011). Emulsifyingproperties of chickpea, faba bean, lentil and pea proteins produced by isoelectric precipitation and salt extraction. Food Research International, 44(9), 2742–2750.

[0011] One of the purposes of the present invention is to solve the deficiencies of existing product technologies and to provide a new method for separating and aggregating proteins in liquids such as human blood.

[0012] The present invention places a mixture of a liquid such as human blood and a diluent in an electrostatic field. The charged nature of proteins in the mixture is exploited to aggregate the proteins on either side of the mixture. If the pH of the liquid is adjusted above or below the isoelectric point of all relevant proteins, the proteins aggregate on one side of the mixture.

[0013] The present invention can precisely control the pH value of the mixture of blood and diluent (with an error of 0.01) by precisely controlling the amount of blood, the pH value and amount of the diluent, and the amount of HCl or NaOH added each time, supplemented by real-time pH measurement and adjustment, thereby achieving the conditions for precipitation and aggregation of various proteins at their respective isoelectric points.

[0014] One of the technical solutions adopted in the present invention is as follows: 1. Place a drop of fresh human venous blood of a defined volume on a glass slide. 2. Add a defined amount of diluent containing HCl to the blood drop on the glass slide, adjusting the pH of the mixture to 2.0. 3. Add varying volumes of NaOH solution to the blood-diluent mixture, gradually increasing the pH to the isoelectric point of each abnormal protein. The pH is then maintained for a set period. During this "pH dwell" period, the voltage applied to the two electrodes is maintained continuously.

[0015] If more than one protein needs to be separated and coagulated at one time, in the above process, the pH value of the mixed solution is increased from small to large, traversing the isoelectric points of all proteins that need to be separated and coagulated, and these proteins are precipitated at their respective isoelectric points.

[0016] To aggregate proteins in a mixture of blood and diluent, the two electrodes—one horizontally ringed and the other vertically cylindrical—are placed. Electric field lines passing through the mixture converge, driving the charged proteins to focus. BRIEF DESCRIPTION OF THE DRAWINGS Instructions attached Figure 1 , is the electric field lines between the two electrodes and their relative position to the mixed liquid.

Claims

1. A method for coagulating protein in a liquid, characterized in that: The pH value of the liquid is increased or decreased, metal electrodes are placed on both sides of the liquid, the electrodes are separated from the liquid by an insulator, and a voltage of 0-900 MegaVolt is applied to the electrodes to cause the proteins in the liquid to aggregate in the area near the electrodes. The pH value of the liquid is adjusted, and the proteins in the liquid undergo isoelectric precipitation.

2. According to claim 1, it is characterized in that The two electrodes are one in a horizontal ring shape and the other in a column shape perpendicular to the horizontal plane.

3. According to claim 1, it is characterized in that The liquid is a mixture of human blood and diluent dropped on a glass slide.

4. According to claim 1, it is characterized in that The insulator is glass and / or air.

5. According to claim 1 and claim 3, it is characterized in that The mixed liquid is a mixed liquid obtained by adding HCl or NaOH aqueous solution to human blood to adjust the pH value.

6. A method for staining and detecting protein aggregation in various human samples, characterized in that: The method for agglomerating proteins in a liquid according to claim 1 to claim 5, or one or more of the claims, is used. The proteins in the human sample include, but are not limited to, PD-L1 protein, p53 protein, Golgi protein 73 (GP73), heat shock protein 90α, abnormal glycan glycoprotein (TAP), central nervous system-specific protein (S100), β2-microglobulin, Alzheimer's-associated neurofilament protein (AD7C-NTP), and albumin.

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