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A blocking-free western blotting method for rapid detection of low-abundance proteins

A western blotting and protein technology, applied in the field of protein detection, can solve the problems of weak protein exposure and development, low sensitivity, and poor effect, and achieve the effects of shortening detection time, avoiding dispersion or escape, and reducing losses

Active Publication Date: 2018-11-27
SOUTH CHINA NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, in general, the proportion of the plant target protein to be analyzed in the total plant protein is relatively low, and the detection effect is often poor by conventional Western blot method, and the protein exposure and development are weak or no development, and the sensitivity is low.

Method used

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  • A blocking-free western blotting method for rapid detection of low-abundance proteins
  • A blocking-free western blotting method for rapid detection of low-abundance proteins
  • A blocking-free western blotting method for rapid detection of low-abundance proteins

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Improved detection of AhNCED protein by western blotting

[0056] Steps (1)-(3) are the same as the above comparative example.

[0057] (4) Carry out methanol treatment to the PVDF membrane that has transferred protein, the concrete method is to let described PVDF membrane soak in 100% methanol for 15s, then let the PVDF membrane treated by methanol carry out natural drying. There are two purposes of drying: to volatilize methanol to eliminate the influence of methanol on subsequent experimental steps; to dry the PVDF membrane to restore the hydrophobic properties of the PVDF membrane. In order not to affect the protein molecules fixed on the PVDF membrane, the PVDF membrane is preferably placed on filter paper to dry naturally.

[0058] Steps (5) and (6) are the same as the above comparative example.

[0059] The development result is as figure 2 As shown, the AhNCED protein in peanut leaves can be seen in the exposure image, but the expression intensity is not as ...

Embodiment 2

[0061] Improved detection of AhNCED protein by western blotting

[0062] Steps (1) to (4) are the same as in the first embodiment above.

[0063] (5) Add the primary antibody (that is, the antibody of the target protein) and incubate at room temperature for 1.5 h, and rinse with TBST washing solution twice, each time for 10 s. Add HRP-labeled secondary antibody (i.e. chromogenic marker) to bind primary antibody, incubate at room temperature for 1 hour, rinse with TBST washing solution twice, each 10s. The implementation of "rinsing" described here is different from the aforementioned "washing", specifically holding a corner of the PVDF membrane with tweezers and gently shaking it in the TBST washing solution in a small range.

[0064] (6) Develop and fix after tableting. This step is the same as the step (6) of the above comparative example.

[0065] The development result is as image 3 As shown, the exposure and development of AhNCED protein in peanut leaves is obvious, ...

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Abstract

The invention discloses a non-closed protein blotting method for quickly detecting low-abundance proteins.The non-closed protein blotting method includes steps of (1), separating total proteins by the aid of gel in a gradient manner; (2), blotting the total proteins separated in the gradient manner on PVDF (polyvinylidene fluoride) films from the gel; (3), soaking the PVDF films with the blotted total proteins in 100% methanol; (4), drying the PVDF films treated by the 100% methanol; (5), carrying out color development detection on the target proteins in the dried PVDF films.The total proteins are extracted from samples.The non-closed protein blotting method for quickly detecting the low-abundance proteins has the advantages that the low-abundance protein detection efficiency can be improved, and practical, sensitive and speedy detection means can be provided.

Description

technical field [0001] The invention relates to a protein detection method, in particular to a non-blocking western blotting method, which is suitable for rapid detection of low-abundance proteins. Background technique [0002] Western blotting (western blot, referred to as WB), also known as immunoblotting (immunoblotting), is a method for detecting a certain protein in a complex sample based on the specific binding of antigens and antibodies. Due to the high resolution of SDS-PAGE and the high specificity and sensitivity of solid-phase immunoassay, western blot has become a common molecular biology technique for protein analysis. [0003] In the process of western blotting, the protein samples separated by PAGE electrophoresis need to be transferred from the PAGE gel to the membrane for fixation. In order to avoid the non-specific binding of the specific antibody of the test reagent to the membrane, it is necessary to carry out the potential binding sites on the membrane. ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/68
CPCG01N33/6803
Inventor 邓斌李玲胡博
Owner SOUTH CHINA NORMAL UNIVERSITY
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