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Goldmag particles based method for immunoprecipitation

A gold magnetic particle, immunoprecipitation technology, applied in biological testing, material testing, etc., can solve the problems of non-specific binding, poor reproducibility, etc., achieve good reproducibility, eliminate the background of non-specific binding, and small loss of activity Effect

Active Publication Date: 2010-06-09
XIAN GOLDMAG NANOBIOTECH
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

[0005] The object of the present invention is to provide a method for immunoprecipitation based on gold magnetic particles, which solves the problems of non-specific binding and poor reproducibility in the existing immunoprecipitation method

Method used

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  • Goldmag particles based method for immunoprecipitation
  • Goldmag particles based method for immunoprecipitation

Examples

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Embodiment 1

[0059] Example 1 is the specific process of separating albumin from serum by gold magnetic particles directly coupled to antibodies:

[0060] 1] Immobilization of human serum albumin antibody on the surface of gold magnetic particles and sealing of gold magnetic particles: Take 4ml of gold magnetic particles into a centrifuge tube with a capacity of 5ml, shake gently and resuspend, place on a magnetic separator, magnetically separate, Discard the supernatant; add 4ml of coupling buffer, shake gently to resuspend, place on a magnetic separator, magnetically separate, discard the supernatant, and repeat the operation once to obtain pretreated gold magnetic particles; human serum albumin antibody Adjust with coupling buffer, prepare a solution with a concentration of 1mg / ml, take 1.5ml and add it to the pretreated gold magnetic particles, react in a shaker at 37°C, 180r / min for 30min, after the reaction is completed, magnetic separation, discard Supernatant; add 4ml of washing bu...

Embodiment 2

[0066] Example 2 is the specific process of separating albumin from serum by immobilized protein A-coupled gold magnetic particles coupled with antibodies:

[0067] 1] Immobilization of protein A on the surface of gold magnetic particles and sealing of gold magnetic particles: Take 1ml of gold magnetic particle suspension and add it to a centrifuge tube with a capacity of 2ml, shake gently to resuspend, place on a magnetic separator, and magnetically separate. Discard the supernatant; add 1ml of coupling buffer (0.2M Tris-HCl buffer at pH 8.0), shake and resuspend, place on a magnetic separator for magnetic separation, discard the supernatant, and repeat the operation once; A is prepared as a solution with a concentration of 1 mg / ml, and 100-200 μl is added to the gold magnetic particles, and reacted in a shaker at 37°C and 180r / min for 30 minutes. After the reaction is completed, the magnetic separation is performed, and the supernatant is discarded; 0.1% Tween-containing PBS...

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Abstract

The invention relates to a goldmag particles based method for immunoprecipitation, belonging to the field of the immunoprecipitation. The goldmag particles based method for immunoprecipitation comprises the following steps of: firstly adding an antibody to a biological sample for immunoprecipitation; and then adding a carrier fixing a protein A / G for separation, or adding a carrier fixing the antibody to the biological sample for the separation after the immunoprecipitation. The method mainly comprises the steps of antibody fixing, immunoprecipitation and elution. The invention solves the problems of nonspecific binding, a few selection varieties of adoptable antibodies and much antibody activity loss in the traditional immunoprecipitation and mainly includes an individual immunoprecipitation (IP) used for separating a known specific protein of the biological sample, a co-immunoprecipitation (Co-IP) used for researching an integral protein complex, testing whether two target proteins are combined in vivo or not and determining the new action cooperation of the specific protein, a chromatin immunoprecipitation (ChIP) used for researching a protein combined with DNA, an RNA immunoprecipitation (RIP) used for researching a protein combined with RNA, and the like.

Description

technical field [0001] The invention relates to a method for performing immunoprecipitation reaction based on magnetic microparticles. Background technique [0002] Immunoprecipitation (Immunoprecipitation, IP) is based on the specific affinity of antibody-antigen, and is used to precipitate or separate and purify the target protein, or to study the interaction between protein, DNA or RNA and the target protein. The principle is: in plasma / clear, cerebrospinal fluid, saliva, ascites, tissue culture, hybridoma supernatant and cell lysate, there are many protein-protein, DNA-protein, RNA-protein interactions, if used If the protein antibody immunoprecipitates the target protein, then the substances that bind to the target protein in vivo can also be precipitated. This method includes individual immunoprecipitation (IP) to isolate known specific proteins in biological samples; to study whole protein complexes, to determine whether two proteins of interest associate in vivo, to...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N33/53
Inventor 崔亚丽胡佳陈超
Owner XIAN GOLDMAG NANOBIOTECH
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