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Horseradish peroxidase black developer, and preparation method and application thereof

A technology of horseradish peroxidase and chromogenic agent, which is applied in the direction of analyzing materials through chemical reactions and analyzing materials through observing the influence of chemical indicators, which can solve the problems of high price and achieve strong contrast , Highly sensitive visual effect, easy to judge the effect of the result

Active Publication Date: 2013-09-25
WENZHOU MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In order to take into account the sensitivity and the final color rendering effect, a well-known foreign company has developed a mixed color developer (DAB / CN) of DAB and CN at present, claiming that the color development is black precipitate, the color development is extremely sensitive, the contrast is strong, and it is convenient for observation, but the actual Applied on film found, expensive and the color, while darker than traditional tans, is still a strong brown

Method used

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  • Horseradish peroxidase black developer, and preparation method and application thereof
  • Horseradish peroxidase black developer, and preparation method and application thereof
  • Horseradish peroxidase black developer, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] The preparation of embodiment 1 black developer

[0031] (1) Prepare separately with water as solvent: 25mg / mL 4-chloro-1-naphthol (4-CN) (note: dissolved in absolute ethanol), 50mg / mL 3,3'-diaminobenzidine (DAB), 50mg / mL nickel ammonium sulfate (note: 0.1M ammonium acetate dissolved), 0.5g / mL cobalt chloride, 0.5g / mL imidazole, 1M boric acid, 20mg / mL MgCl2, 20mg / mLMnCl2, 25mg / mL nitrosoferricyanide Sodium, 12 mg / mL Betaine, 2 mg / mL Gelatin, 0.2 g / mL Dextran Sulfate, 1% (V / V) THF, 1% (V / V) Acetone.

[0032] (2) Take 25mg / mL 4-CN 2.5μL, 50mg / mL DAB 2.5μL, 50mg / mL nickel ammonium sulfate 5μL, 0.5g / mL cobalt chloride 0.5μL, 0.5g / mL imidazole 0.5μL, 1M boric acid 1μL, 20mg / mL MgCl 21 μL, 20 mg / mL MnCl 21 μL, 25 mg / mL sodium nitroferricyanide 1 μL, 12 mg / mL betaine 1 μL, 2 mg / mL gelatin 1 μL, 0.2 g / mL dextran sulfate 1 μL, 1% (V / V ) THF 1 μL, 1% (V / V) acetone 1 μL, polyethylene glycol octylphenyl ether (Triton X-100) 1 μL, 30wt% H2O2 1 μL, 1×TBS (prepared according to "M...

Embodiment 2

[0033] Example 2 Membrane Detection of Synthetic Nucleic Acid Molecules

[0034] (1) RNA molecule miR-29b was synthesized by GenePharma; a biotin-labeled DNA probe (antisense sequence of miR-29b) was synthesized by Shanghai Boshang Biotechnology Co., Ltd.

[0035] (2) Take 10 pmol of the above-mentioned synthesized RNA and DNA probes and operate according to the steps of LHCD (liquid phase hybridization chromogenic method) (X.Li, M.Ni, and Y.Zhang, Methods85(2), 151(2012)) , where the spotting was repeated for three membranes.

[0036] (3) Different color-developing solutions (DAB, DAB / CN and the black color-developing agent prepared in Example 1) were used for color development for 20 seconds, and then water was used to stop the color development.

[0037] The result is as figure 1 As shown, A: DAB, B: DAB / CN (purchased from Thermo scientific), C: black color reagent prepared in Example 1; 1: no probe, 2: no RNA, 3: irrelevant RNA (Gimma Synthetic General Control), 4: add ...

Embodiment 3

[0038] Example 3 Membrane Detection of Synthetic Protein Molecules

[0039] (1) The FLAG TAG peptide was synthesized by Gill Biochemical Co., Ltd.

[0040] (2) Spot 50ng of the protein from step (1) on the nylon membrane, repeat the spotting three times, and air-dry.

[0041] (3) Block the membrane with 10% BSA (bovine serum albumin) at a mass volume ratio for 1 hour at room temperature.

[0042] (4) Hybridize with biotin-labeled rabbit anti-FLAG TAG antibody (purchased from Shanghai Boyao Biological Reagent Co., Ltd.) for 1 hour at room temperature.

[0043] (5) Wash with TBST (prepared according to "Molecular Cloning") for 3 times, 10 minutes each time.

[0044] (6) Amplify the signal with ABC (avidin-biotin complex) and then wash the membrane according to LHCD (liquid phase hybridization color method) (X.Li, M.Ni, and Y.Zhang, Methods85 (2 ), 151(2012)).

[0045] (7) Different color-developing solutions (DAB, DAB / CN and the black color-developing agent prepared in Examp...

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Abstract

The invention relates to a horseradish peroxidase black developer, and a preparation method and an application thereof, and belongs to the technical field of biology. The raw materials of the horseradish peroxidase black developer comprise: 4-CN, DAB, ammonium nickel sulfate, cobaltous chloride, imidazole, polyoxyethylene octyl phenyl ether, boric acid, MgCl2, MnCl2, sodium nitroferricyanide, betaine, gelatin, tetrahydrofuran, acetone, dextran sulfate, 1*TBS and H2O2. The horseradish peroxidase black developer is obtained by uniformly mixing a certain amount of the raw materials. By using the horseradish peroxidase black developer, the color development of a horseradish peroxidase has the advantages of high sensitivity of DAB and good color-difference visual sense effect of 4-CN at the same time, and the color substance is a black deposition with a strong background contrast, and therefore the result determine is convenient.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a horseradish peroxidase black color developer and a preparation method thereof and its application in the field of nucleic acid / protein detection. Background technique [0002] Peroxidase labeling and alkaline phosphatase (AP) labeling chromogenic technology are two basic technologies for nucleic acid and protein detection. Among them, the peroxidase marker is the most commonly used marker. The peroxidase is generally horseradish peroxidase (HRP). The reason for this name is that HRP is obtained from a plant called horseradish. The roots are isolated and purified. HRP has the following advantages: (1) small molecule (molecular mass 40kDa), small steric barrier, strong penetrating power, and easy binding to intracellular antigens; (2) stable properties and easy access to high-purity enzyme products; ( 3) High activity and strong specificity; (4) The enzyme reaction prod...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/78
Inventor 李湘麒谭毅张弛梁广
Owner WENZHOU MEDICAL UNIV
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