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Detection method of WAS protein

A detection method and protein technology, applied in the field of WAS protein detection, can solve the problems of long time and complicated process, and achieve the effects of simple steps, saving consumption, and being suitable for wide application.

Pending Publication Date: 2021-08-13
CHILDRENS HOSPITAL OF CHONGQING MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] This application provides a method for detecting WAS protein to solve the problem that it is necessary to separate PBMC from peripheral blood to detect WAS protein, resulting in a complicated and time-consuming process

Method used

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  • Detection method of WAS protein
  • Detection method of WAS protein
  • Detection method of WAS protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0085] 1) Prepare 2 flow tubes, marked as Test (sample to be tested) and HC (normal sample), add 50ul of the sample to be tested, 50ul of whole blood of the normal sample, 2ul of various fluorescently labeled antibodies and Fixable Viability Dye dye 0.2ul, vortex mixed, fixed at 4°C for more than 30min;

[0086] 2) Then add 0.5-1.5ml 1×PBS, vortex and mix, discard the supernatant after centrifugation, then add 500ul of fixative, vortex and mix, fix at 3-5℃ for more than 30min;

[0087] 3) Discard the supernatant fixative after centrifugation (3500rpm, 3min, temperature is not limited), and keep the cells at the bottom of the tube;

[0088] 4) Add 1ml 1×PBS (phosphate buffer saline) respectively, vortex and mix well, centrifuge (3500rpm, 3min, temperature is not limited) and discard the supernatant;

[0089] 5) Add 5ml of erythrocyte lysate respectively, blow and mix with a pipette tip, and treat in a 37°C water bath for 4-6 minutes until the liquid in the flow tube turns transp...

Embodiment 2

[0106] 1) Prepare 2 flow tubes, marked as Test (sample to be tested) and HC (normal sample), respectively add the sample to be tested, 40ul of whole blood of the normal sample, add 1ul of various fluorescently labeled antibodies and Fixable Viability Dye dye 0.1ul, vortex mixed, fixed at 4°C for more than 30min;

[0107] 2) Then add 0.5ml 1×PBS, vortex and mix, discard the supernatant after centrifugation, then add 400ul of fixative, vortex and mix, fix at 3°C ​​for more than 30min;

[0108] 3) Discard the supernatant fixative after centrifugation (3500rpm, 3min, temperature is not limited), and keep the cells at the bottom of the tube;

[0109] 4) Add 1ml 1×PBS (phosphate buffer saline) respectively, vortex and mix well, centrifuge (3500rpm, 3min, temperature is not limited) and discard the supernatant;

[0110] 5) Add 5ml of erythrocyte lysate respectively, blow and mix with a pipette tip, and treat in a 37°C water bath for 4-6 minutes until the liquid in the flow tube turn...

Embodiment 3

[0121] 1) Prepare 2 flow tubes, marked as Test (sample to be tested) and HC (normal sample), add the sample to be tested, 60ul of whole blood of the normal sample, 3ul of various fluorescently labeled antibodies and Fixable Viability Dye dye 0.3ul, vortex mixed, fixed at 4°C for more than 30min;

[0122] 2) Then add 1.5ml 1×PBS, vortex mix, discard the supernatant after centrifugation, then add fixative 400-600ul, vortex mix, fix at 3-5℃ for more than 30min;

[0123] 3) Discard the supernatant fixative after centrifugation (3500rpm, 3min, temperature is not limited), and keep the cells at the bottom of the tube;

[0124] 4) Add 1ml 1×PBS (phosphate buffer saline) respectively, vortex and mix well, centrifuge (3500rpm, 3min, temperature is not limited) and discard the supernatant;

[0125] 5) Add 5ml of erythrocyte lysate respectively, blow and mix with a pipette tip, and treat in a 37°C water bath for 4-6 minutes until the liquid in the flow tube turns transparent;

[0126] ...

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Abstract

The invention relates to a WAS protein detection method, which comprises: taking 400-600 [mu]l of a fixing agent, mixing with 40-60 [mu]l of peripheral blood of a to-be-detected sample, and fixing for more than 30 min at a temperature of 3-5 DEG C to obtain a first product; centrifuging, discarding the fixing agent in the first product, washing with a 1*phosphate buffer solution, mixing with a red blood cell lysis solution, putting into a water bath kettle at 36.5-37.5 DEG C for 4-6 minutes to lyse red blood cells, adding a punching agent, and breaking a membrane and punching for 14-16 minutes at room temperature to obtain a second product; mixing the second product with a mouse anti-human WAS protein IgG2alpha antibody, and incubating at room temperature for 30 minutes; and eluting the uncombined free antibody, mixing with the rat anti-mouse IgG2alpha antibody, incubating at room temperature for 30 minutes, washing, detecting on a flow machine to obtain detection data, and analyzing the detection data. According to the method, the WAS protein level can be directly detected in the peripheral blood, the step of separating the PBMC from the peripheral blood is omitted, and the use amount of the to-be-detected sample is reduced; the method is simple and rapid in steps, and is more suitable for wide application in WAS protein detection.

Description

technical field [0001] The present application relates to the technical field of WAS protein detection, in particular to a detection method of WAS protein. Background technique [0002] WAS protein (Wiskott-Aldrich Syndrome protein) is specifically expressed in the hematopoietic system, and is a key regulator of cytoskeleton and immune synapse formation. essential. [0003] WAS protein is specifically expressed in other immune cells except red blood cells; the subcellular localization of WAS protein is mainly in the cytoplasm, and a small amount is in the nucleus and cell membrane. [0004] Lymphocytes are an important class of immune cell lines produced by lymphoid organs. According to their phenotype and functional characteristics, they can be divided into different categories, such as T cells, B cells, NK cells, etc. Lymphocytes cooperate and restrict each other in the process of immune response, and jointly complete the recognition, response and clearance of antigenic...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/58G01N21/64
CPCG01N33/68G01N33/582G01N21/6428G01N21/6486G01N2021/6439G01N2333/4703
Inventor 赵晓东罗贤泽周丽娜
Owner CHILDRENS HOSPITAL OF CHONGQING MEDICAL UNIV