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Immunofluorescence labeling method for proteins in living cells

An immunofluorescence and labeling method, applied in the field of immunofluorescence labeling of proteins in living cells, can solve the problems of complex technical procedures, inability to label living cell proteins, lack of objectivity, etc., and achieve the effect suitable for popularization and application.

Pending Publication Date: 2019-10-15
陈彩丽
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The main disadvantages are: the problem of non-specific staining has not been completely resolved, the objectivity of the result judgment is insufficient, and the technical procedures are still relatively complicated
[0006] The immunofluorescent labeling method in the prior art can only label fixed cells (damage live cells first and then label), but cannot label proteins in live cells, so this method cannot be used to observe the dynamic changes of live cells. for further biological research

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] A method for immunofluorescent labeling of proteins in living cells, comprising the following steps;

[0018] The cell smear was treated with 0.2% TritonX-100 for 10 min, washed with 0.01 MPBS for 3 min, washed 3 times, digested with 0.01 trypsin solution at 37°C for 12 min, washed with 0.01 MPBS for 3 min, washed 3 times, and blocked with normal rabbit serum. Add FITC-goat anti-mouse IgG, incubate at 37°C for 30 minutes, wash with 0.01MPBS for 3 minutes, use BrdU as a protein marker, fix with cross-linking fixative, and observe under a fluorescence microscope.

[0019] The cross-linking fixative contains: acetone, paraformaldehyde and PBS buffer.

[0020] The fluorescence microscope observation adopts a laser scanning confocal microscope.

Embodiment 2

[0022] A method for immunofluorescent labeling of proteins in living cells, comprising the following steps;

[0023] The cell smear was treated with 0.2% TritonX-100 for 12 minutes, washed with 0.01 MPBS for 4 minutes, washed 4 times, digested with 0.01 trypsin solution at 38°C for 13 minutes, washed with 0.01 MPBS for 4 minutes, washed 4 times, and blocked with normal rabbit serum. Add FITC-goat anti-mouse IgG, incubate at 38°C for 35 minutes, wash with 0.01MPBS for 4 minutes, use BrdU as a protein marker, fix with cross-linking fixative, and observe under a fluorescence microscope.

[0024] The cross-linking fixative contains: acetone, paraformaldehyde and PBS buffer.

[0025] The fluorescence microscope observation adopts a laser scanning confocal microscope.

Embodiment 3

[0027] A method for immunofluorescent labeling of proteins in living cells, comprising the following steps;

[0028] The cell smear was treated with 0.2% TritonX-100 for 15 minutes, washed with 0.01 MPBS for 5 minutes, washed 5 times, digested with 0.01 trypsin solution at 40°C for 15 minutes, washed with 0.01 MPBS for 5 minutes, washed 5 times, and blocked with normal rabbit serum. Add FITC-goat anti-mouse IgG, incubate at 40°C for 40 minutes, wash with 0.01MPBS for 5 minutes, use BrdU as a protein marker, fix with cross-linking fixative, and observe under a fluorescence microscope.

[0029] The cross-linking fixative contains: acetone, paraformaldehyde and PBS buffer.

[0030] The fluorescence microscope observation adopts a laser scanning confocal microscope.

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Abstract

The present invention discloses an immunofluorescence labeling method for proteins in living cells, and relates to the field of biotechnology. The method comprises following steps: a cell smear is treated through 0.2% TritonX-100 for 10-15 minutes, is washed with 0.01 M PBS for 3-5 minutes, is washed for 3-5 times, is digested with a 0.01 trypsin solution at 37-40 degree Celsius for 12-15 min, iswashed with the 0.01 M PBS for 3 - 5 minutes, is washed for 3 - 5 times, then is blocked with normal rabbit serum, is added with FITC-goat-anti-mouse iGg, is incubated at 37-40 degree Celsius for 30-40 minutes, is washed with the 0.01 M PBS for 3 - 5 minutes, with BrdU used as a protein marker, and after fixed with cross-linking fixative, is observed under a fluorescence microscope. The method canobserve and detect a morphological structure, molecular and ion number of the living cells in real time, can simultaneously study a plurality of target proteins on one specimen, and is suitable for popularization and application.

Description

technical field [0001] The invention belongs to the field of biotechnology and relates to an immunofluorescence labeling method for proteins in living cells. Background technique [0002] Immunofluorescence technique (Immunofluorescence technique), also known as fluorescent antibody technology, is one of the earliest developed labeling immune techniques. It is a technique established on the basis of immunology, biochemistry and microscopy. For a long time, some scholars have attempted to combine antibody molecules with some tracer substances, and use antigen-antibody reactions to locate antigenic substances in tissues or cells. [0003] The main features of this technique are: strong specificity, high sensitivity and fast speed. The main disadvantages are: the problem of non-specific staining has not been completely resolved, the objectivity of the result judgment is insufficient, and the technical procedures are still relatively complicated. [0004] Fluorescence immunoa...

Claims

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Application Information

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IPC IPC(8): G01N33/533
CPCG01N33/533
Inventor 陈彩丽
Owner 陈彩丽
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