Reagent, preparation method and application for separating and extracting plasma protein and nucleoprotein in cells

A nuclear protein and cytoplasmic technology, applied in the field of protein separation, can solve problems such as cross-contamination, poor storage stability, and reagent toxicity, and achieve the effects of avoiding mutual contamination, improving storage stability, and reducing cytotoxicity

Active Publication Date: 2022-03-01
JIANGXI PROVINCIAL PEOPLES HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In view of the above situation, the present invention provides a reagent, preparation method and application for separating and extracting plasma protein and nucleoprotein in cells, so as to solve the problem of cross-contamination and strong toxicity of the reagents when the existing reagents separate cytoplasmic protein and nucleoprotein. , The problem of poor storage stability

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  • Reagent, preparation method and application for separating and extracting plasma protein and nucleoprotein in cells

Examples

Experimental program
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Effect test

Embodiment 1

[0088]A reagent for separating plasma protein and nuclear protein in cells, including lysate A, lysate B and lysate C, the composition of lysate A is: 10mM HEPES with pH 7.8, 10mM NaCl, 0.5 mM EDTA, 1.5mM MgCl 2 ;The composition of lysate B is: 10mM HEPES at pH 7.8, 10mM NaCl, 0.5 mM EDTA, 1.5mM MgCl 2 , 10% NP-40; the composition of lysate C is: 10mM HEPES at pH 7.8, 10mM NaCl, 0.5 mM EDTA, 1.5mM MgCl 2 , 10% glycerin, 10% SDS.

[0089] The preparation method of the reagent for separating and extracting plasma protein and nucleoprotein in cells includes:

[0090] Step 1: prepare lysate A;

[0091] Step 2: After obtaining lysate A, prepare lysate B on the basis of lysate A;

[0092] Step 3: After obtaining lysate A, prepare lysate C on the basis of lysate A.

[0093] Wherein, the step 1 specifically includes:

[0094] Step 1.1: Prepare HEPES solution with a concentration of 10mM;

[0095] Step 1.2: Prepare NaCl and MgCl 2 Two salt solutions, the concentration of NaCl i...

Embodiment 2

[0102] A reagent for separating plasma protein and nuclear protein in cells, including lysate A, lysate B and lysate C, the composition of lysate A is: 6mM HEPES with pH 7.8, 10mM NaCl, 0.5 mM EDTA, 2mM MgCl 2 ; The composition of lysate B is: 6 mM HEPES at pH 7.8, pH 7.8, 10 mM NaCl, 0.5 mM EDTA, 2 mM MgCl 2 , 12% NP-40; the composition of lysate C is: pH 7.8 6mM HEPES, 10mM NaCl, 0.5 mM EDTA, 2mM MgCl 2 , 10% glycerin, 9% SDS.

[0103] The preparation method of the reagent for separating and extracting plasma protein and nucleoprotein in cells includes:

[0104] Step 1: prepare lysate A;

[0105] Step 2: After obtaining lysate A, prepare lysate B on the basis of lysate A;

[0106] Step 3: After obtaining lysate A, prepare lysate C on the basis of lysate A.

[0107] Wherein, the step 1 specifically includes:

[0108] Step 1.1: Prepare HEPES solution with a concentration of 6mM;

[0109] Step 1.2: Prepare NaCl and MgCl 2 Two salt solutions, the concentration of NaCl is ...

Embodiment 3

[0116] A reagent for separating plasma protein and nuclear protein in cells, including lysate A, lysate B and lysate C, the composition of lysate A is: 9mM HEPES with pH 7.8, 11mM NaCl, 0.5 mM EDTA, 1mM MgCl 2 ;The composition of lysate B is: 9mM HEPES at pH 7.8, pH 7.8, 11mM NaCl, 0.5 mM EDTA, 1mM MgCl 2 , 11% NP-40; the composition of lysate C is: pH 7.8 9mM HEPES, 11mM NaCl, 0.5 mM EDTA, 1mM MgCl 2 , 12% glycerin, 9% SDS.

[0117] The preparation method of the reagent for separating and extracting plasma protein and nucleoprotein in cells includes:

[0118] Step 1: prepare lysate A;

[0119] Step 2: After obtaining lysate A, prepare lysate B on the basis of lysate A;

[0120] Step 3: After obtaining lysate A, prepare lysate C on the basis of lysate A.

[0121] Wherein, the step 1 specifically includes:

[0122] Step 1.1: Prepare HEPES solution with a concentration of 9mM;

[0123] Step 1.2: Prepare NaCl and MgCl 2 Two salt solutions, the concentration of NaCl is 11mM...

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Abstract

The invention provides a reagent, preparation method and application for separating and extracting plasma protein and nucleoprotein in cells. The reagent includes lysate A: 6mM-12mM HEPES with a pH of 7.8, 9mM-11mM NaCl, 0.5 mM EDTA, 1mM- 2mM MgCl 2 ; Lysis Buffer B: 6mM‑12mM HEPES pH 7.8, pH 7.8, 9mM‑11mM NaCl, 0.5 mM EDTA, 1mM‑2mM MgCl 2 , 8%‑12% NP‑40; Lysis Solution C: 6mM‑12mM HEPES, pH 7.8, 9mM‑11mM NaCl, 0.5 mM EDTA, 1mM‑2mM MgCl 2 , 8%-12% glycerin, 8%-12% SDS. The invention can realize efficient, rapid and complete separation of cytoplasmic protein and nucleoprotein, and avoid cross contamination of the two.

Description

technical field [0001] The invention relates to the technical field of protein separation, in particular to a reagent, preparation method and application for separating and extracting plasma protein and nucleoprotein in cells. Background technique [0002] Nucleoplasmic protein separation reagent is a reagent used to extract proteins in cells. Cell proteins commonly used in medical research are cytoplasmic proteins and nuclear proteins; due to the high intracellular osmotic pressure, a lysate with low osmotic pressure is usually used to destroy the cell membrane. When the cell membrane is lysed, the cytoplasmic protein will leak out; high salt can damage the cell In the high-salt environment, the nuclear membrane is destroyed, and the nuclear protein can be released; therefore, the key to the separation technology of nuclear plasma protein is to effectively separate the cytoplasm and nuclear protein, namely plasma protein, by using reagents with different osmotic pressures. ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/00C07K1/14C12N5/00
CPCC07K14/00C12N5/00C12N2501/998
Inventor 李蓉
Owner JIANGXI PROVINCIAL PEOPLES HOSPITAL
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