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Protein immunodetection method

An immune detection method and protein technology are applied in the field of biological detection to achieve the effects of improving signal-to-noise ratio, strong versatility and saving usage.

Inactive Publication Date: 2017-02-22
苏州拜恩翰斯生物医药有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] Based on this, it is necessary to provide a protein immunoassay method with reduced noise amplification for the simultaneous amplification of noise in protein immunoassay methods

Method used

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Experimental program
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Embodiment 1

[0068] A549 cells (human non-small cell lung cancer cells) were cultured in high-glucose DMEM medium with 10% fetal bovine serum at 37°C, 5% CO 2 Cultured in an incubator, subcultured once every 3 days, and the cells in the logarithmic growth phase were taken for experiments.

[0069] Place a plate (9cm) of cells (approximately 10 7 ) was lysed in 1 mL RIPA lysate (50 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS). And incubate on ice for 20 minutes, then centrifuge at 12000r / min at 4°C for 15 minutes to obtain the total protein.

[0070] The protein concentration was measured by BCA method and aliquoted.

[0071] Take 20 μg of total protein per well, separate by 12% polyacrylamide gel electrophoresis, transfer to PVDF membrane, block with 5% (W / W) skimmed milk for 1 hour, and then cut the membrane into two parts.

[0072] Add 1:5000 diluted α-Tubulin primary antibody to one of the membranes, incubate overnight at 4°C, wash the membrane wi...

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Abstract

The invention relates to the technical field of biological detection, in particular to a protein immunodetection method. The method includes the following steps that target protein is transferred to a solid phase carrier; sealing is carried out; primary antibodies are added, and inoculation washing is carried out; engineering enzymes are added, and inoculation washing is carried out; adenosine triphosphoric acid and magnesium ions are added, and inoculation washing is carried out; peroxidase is added, and inoculation washing is carried out; a chemiluminescence reagent is added for imaging and developing. According to the method, based on the new signal enhancement principle, the target protein can be modified through an enzymatic reaction, signal sources are increased, the specificity and reactivity of the primary antibodies are selectively enhanced, noise is not amplified while signals are enhanced accordingly, and then the signal to noise ratio is effectively increased. The primary antibodies are saved, and cost is reduced. Peroxidase is independently used, high universality is achieved, it is not needed to select different secondary antibodies for primary antibodies in mouse sources or rabbet sources, and therefore the method is economical and practical. The method can be suitable for WB and ELISA.

Description

technical field [0001] The invention relates to the technical field of biological detection, in particular to a protein immune detection method. Background technique [0002] Western Blotting technology (Western blotting technology) is a method of separating protein samples by polyacrylamide gel electrophoresis, transferring them to a solid-phase carrier, and using the specific binding reaction of antigens and antibodies to detect the expression or modification state of the target protein. Western Blotting technology is currently the most commonly used method for detecting proteins in biochemical experiments. However, this method often encounters problems such as too high background and too weak signal, which affects the analysis of experimental results and the progress of experiments. [0003] Factors such as the abundance of the target protein (i.e., antigen) in the total protein, the specificity and reactivity of antibody binding, and the control of the signal-to-noise ra...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68
CPCG01N33/6803
Inventor 刘华东
Owner 苏州拜恩翰斯生物医药有限公司