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ECL substrate liquid and preparation method and application thereof

A technology of substrate solution and biological preservative, applied in the biological field, can solve the problems of phosphorylated antibodies that cannot achieve good results, no specific phosphorylated protein application, low phosphorylated protein content, etc., to reduce background value and cost Low, enhanced substrate fluorescence effect

Active Publication Date: 2018-12-25
ELABSCI BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the content of phosphorylated protein is relatively low. In the process of WB detection, the expression abundance of conventional luminol substrate solution is often low and cannot be detected. This is mainly due to the use of conventional luminol substrate The sensitivity of the solution is relatively low, and there is no specific application for the detection of phosphorylated proteins
That is, the sensitivity of the conventional ECL substrate solution is low, and it cannot achieve good results in the detection of phosphorylated antibodies.

Method used

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  • ECL substrate liquid and preparation method and application thereof
  • ECL substrate liquid and preparation method and application thereof
  • ECL substrate liquid and preparation method and application thereof

Examples

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Effect test

Embodiment 1

[0029] The present embodiment provides a kind of ECL substrate liquid, comprises the component of following concentration: the 1-bromo-2-naphthol of 0.30g / L, the 1-bromo-2-naphthol of 0.28g / L, the EDTA of 0.50g / L, 0.10g / L of carbamide peroxide, 2.00g / L of polyethylene glycol 6000, 0.20mL / L of Proclin300 and 0.40mol / L of pH 9.0 citric acid buffer.

[0030] The preparation method of the ECL substrate liquid of the present embodiment comprises the steps:

[0031] Preparations: Clean the 1000mL graduated cylinder and 1000mL volumetric flask with tap water, rinse the stirring bar for later use, balance the analytical balance and preheat it for 20 minutes, and calibrate the pH meter.

[0032] Use weighing paper to accurately weigh 5.5g of citric acid on an analytical balance, and slowly pour it into a dried 1000mL beaker.

[0033] Use weighing paper to accurately weigh 14.5g of sodium citrate on an analytical balance, and slowly pour it into the above-mentioned dried 1000mL beaker...

Embodiment 2

[0042] The ECL substrate solution prepared in Example 1 was applied to WB detection of phosphorylated P53, and the specific steps were as follows:

[0043] S1, cell treatment:

[0044] Place the HCT116 cell lysate cell culture on a petri dish, and put ice under the petri dish. The HCT116 cell lysate cell culture was washed with an iced (temperature range of 0-4° C., the same below) PBS solution. Aspirate the PBS solution, then add ice-cold lysis buffer.

[0045] Scrape the lysed cells from the culture dish with an ice-cold plastic cell scraper and place at 4°C for 30 min with continuous stirring. Centrifuge again at 4°C (different cell lysis requires different centrifugal force and time). Gently remove the centrifuge tube from the centrifuge, absorb the supernatant, transfer to a new pre-cooled centrifuge tube, and remove particulate matter to obtain the lysate.

[0046] S2, use the BCA method to measure the standard curve, and calculate the loading amount:

[0047] Make ...

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Abstract

The invention relates to an ECL substrate liquid and a preparation method and an application thereof. The ECL substrate liquid includes the following components: luminol 0.15g / L-0.50g / L, 1-bromo-2-naphthol 0.15g / L-0.50g / L, EDTA 0. 15g / L-1.00g / L, urea peroxide 0.03g / L-0.20g / L, polyethylene glycol 1.00g / L-5.00g / L, biological preservative 0.05mL / L- 0.50 mL / L and citrate buffer with a pH of 9.0 0.2 mol / L-0.6 mol / L. The ECL substrate liquid is formed by the combination of specific components to form a luminescent liquid for Western blotting detection, which can enhance the fluorescence of the substrate, and can greatly improve the detection sensitivity, reduce the background value, and heat during the Western blotting experiment, has good thermal stability and can be stored for a long time.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an ECL substrate liquid and its preparation method and application. Background technique [0002] Western blotting (WB for short) is a hybridization technique that combines high-resolution gel electrophoresis and immunochemical analysis techniques. Western blotting has the advantages of large analysis capacity, high sensitivity, and strong specificity. It is the most commonly used method for detecting protein characteristics, expression, and distribution, such as qualitative and quantitative detection of tissue antigens, mass determination of polypeptide molecules, and detection of viruses. Antibody or antigen detection, etc. [0003] Protein phosphorylation refers to the process of transferring the phosphate group of adenosine triphosphate (ATP) to amino acid residues (serine, threonine, tyrosine) of the substrate protein catalyzed by protein kinases, or binding to guanosine tripho...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/533G01N33/561G01N33/68G01N33/53
CPCG01N33/5306G01N33/533G01N33/561G01N33/6854G01N2333/4748
Inventor 冷毅斌张念元
Owner ELABSCI BIOTECH