Preparation method of protein powder used for blocking transfer membrane in protein immunoblotting, and blocking method for protein immunoblotting

A technology of western blotting and protein, applied in the field of protein research, can solve the problems of low protein content of skimmed milk powder, pollution, interference, etc., achieve the effect of clean exposure background, avoid background reaction, and enhance specificity

Pending Publication Date: 2020-04-10
莫纳(武汉)生物科技有限公司
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Problems solved by technology

The disadvantage is that the protein content of skim milk powder is not high. Compared with the protein content of BSA as high as 90%, the protein content of skim milk powder is only more than 30%. Moreover, the protein content of skim milk powder is still mainly derived from mammals. In the homologous WB experiment, it is possible cause disturbance or pollution

Method used

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  • Preparation method of protein powder used for blocking transfer membrane in protein immunoblotting, and blocking method for protein immunoblotting
  • Preparation method of protein powder used for blocking transfer membrane in protein immunoblotting, and blocking method for protein immunoblotting
  • Preparation method of protein powder used for blocking transfer membrane in protein immunoblotting, and blocking method for protein immunoblotting

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Embodiment 1, a kind of preparation method of the fish gelatin protein powder that is used for sealing transfer membrane in western blotting, its steps are as follows:

[0052] (1) Dissolve marine fish skin gelatin with PBS buffer solution to prepare a fish skin gelatin solution with a mass concentration of 15%;

[0053] (2) Prepare bovine trypsin and thrombin freeze-dried powder in a weight ratio of 3:1, and use sterilized purified water to prepare a 1 mg / mL mixed enzyme solution for later use;

[0054] (3) Keep the prepared fish gelatin solution at constant temperature to 37°C and stir at 80rpm;

[0055] (4) Slowly add an appropriate amount of the prepared mixed enzyme solution, and incubate at 37°C for 6 hours;

[0056] (5) Raise the temperature to 60°C and keep it warm for 30 minutes to inactivate the enzyme and end the enzymatic hydrolysis reaction;

[0057] (6) Treat the reaction solution with a two-stage ultrafiltration membrane system. The first-stage membrane...

Embodiment 2

[0059] Embodiment 2, a kind of preparation method of the fish gelatin protein powder that is used for sealing transfer membrane in western blotting, its steps are as follows:

[0060] (1) Dissolve marine fish skin gelatin with PBS buffer solution to prepare a fish skin gelatin solution with a mass concentration of 25%;

[0061] (2) Prepare bovine trypsin and thrombin freeze-dried powder in a ratio of 2:1 by weight, and use sterilized purified water to prepare a 1 mg / mL mixed enzyme solution for later use;

[0062] (3) Keep the prepared fish gelatin solution at a constant temperature to 37°C and stir at 100rpm;

[0063] (4) Slowly add an appropriate amount of the prepared mixed enzyme solution, and incubate at 37°C for 6 hours;

[0064] (5) Raise the temperature to 60°C and keep it warm for 30 minutes to inactivate the enzyme and end the enzymatic hydrolysis reaction;

[0065] (6) Treat the reaction solution with a two-stage ultrafiltration membrane system. The first-stage me...

Embodiment 3

[0067] Embodiment 3, a kind of western blotting blocking method, this method uses the fish gelatin protein powder obtained in embodiment 1 or 2 as blocking solution raw material, and its specific steps are as follows:

[0068] (1) Extraction of protein samples: take freshly cultured A549 cells, remove the culture medium, and wash the cells with PBS; add 200 μl RIPA cell lysate to each well of a 6-well plate, containing a final concentration of 1 mM PMSF, and blow it several times with a gun. Make the lysate fully contact with the cells and lyse the cells; centrifuge the lysed cell samples at 14,000g for 5min, take the supernatant and put them in PCR tubes, and store them at -80℃ for later use;

[0069] (2) Protein quantification and sample loading pre-treatment: take the protein extract prepared in step (1) and use the BCA method to determine the protein concentration; Mix well and place in a 95°C metal bath for denaturation for 10 minutes, then place in a transient state at -...

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Abstract

The invention relates to a preparation method of fish gelatin protein powder for blocking a transfer membrane in protein immunoblotting. The method comprises the following steps: dissolving marine fish skin gelatin by using a PBS buffer solution to prepare a fish skin gelatin solution, carrying out enzymolysis by using a mixed enzyme solution of bovine trypsin and thrombin freeze-dried powder, andcarrying out ultrafiltration, enriching, and drying successively to obtain the fish gelatin protein. The invention also discloses a blocking method for protein immunoblotting. According to the invention, the common problems of poor blocking effect, high exposure background, non-specific strip detection and the like of a transfer printing film in a protein immunoblotting experiment are effectivelysolved. By adopting a blocking agent for an immunoblotting experiment in the invention, a specific protein detection zone can be obtained through ECL substrate detection after antibody incubation, the background is clean and free of impurity zones, the incubation time is shortened to 15 min, and a technical basis is provided for obtaining accurate and efficient experiment results.

Description

technical field [0001] The invention belongs to the technical field of protein research, and in particular relates to a preparation method of fish gelatin protein powder used for sealing transfer membranes in western blotting of proteins; the invention also discloses a sealing method of western blotting of proteins. Background technique [0002] Western Blot technology is a method in which protein samples are separated by polyacrylamide gel electrophoresis, transferred to a solid phase support (such as nitrocellulose membrane), and the target protein is detected by antigen-antibody reaction. It is an experimental method commonly used in molecular biology, biochemistry and immunogenetics. [0003] The conventional Western Blot operation process includes: protein sample preparation, electrophoretic separation, membrane transfer, blocking, washing, primary antibody incubation, washing, secondary antibody incubation, washing, and detection. However, this method involves many st...

Claims

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Application Information

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IPC IPC(8): C12P21/06C07K14/78C07K1/34G01N33/53
CPCC12P21/06C07K14/78G01N33/5306
Inventor 葛绍勇赵盼盼刘杨陈志强夏加权姜林先
Owner 莫纳(武汉)生物科技有限公司
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