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Transfer buffer for promoting residence of small molecular proteins on PVDF membrane

A small molecule protein, membrane transfer technology, applied in analytical materials, measuring devices, instruments, etc., can solve the problems of not supporting small molecule protein interception, unable to achieve accurate interception, small molecule protein escape, etc., and achieve a simple detection method. Efficient, not easy to transfer membrane failure, improve the effect of protein retention rate

Inactive Publication Date: 2019-08-13
INST OF HEMATOLOGY & BLOOD DISEASES HOSPITAL CHINESE ACADEMY OF MEDICAL SCI & PEKING UNION MEDICAL COLLEGE
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Problems solved by technology

The various transfer buffers mentioned above have been verified by experiments, none of which support the retention of small molecular proteins on 0.45 μM PVDF, and only a short transfer time and a low transfer current can be used. Once the transfer time is slightly extended or the transfer A slight increase in the membrane current will lead to the escape of small molecular proteins, which cannot achieve precise interception, which brings a great error factor to the experiment.

Method used

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  • Transfer buffer for promoting residence of small molecular proteins on PVDF membrane
  • Transfer buffer for promoting residence of small molecular proteins on PVDF membrane
  • Transfer buffer for promoting residence of small molecular proteins on PVDF membrane

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] A method for detecting small molecular proteins, the main steps are as follows:

[0048] 1. Cut out the 0.45μM PVDF membrane (approximately 5cm×8.5cm) covering the whole gel, soak in 20mL isopropanol for 30s, shake to completely infiltrate;

[0049] 2. Discard isopropanol, add 40mL of 10% isopropanol prepared transfer solution, shake and mix well to avoid precipitation of glycine;

[0050] 3. Discard the liquid, add 40mL of transfer solution prepared with 10% isopropanol again, shake and mix well to avoid precipitation of glycine;

[0051] 4. Pour off the liquid, add 40mL 10% isopropanol prepared transfer solution for the third time, vortex and mix well for later use;

[0052] 5. Use the transfer buffer prepared with 10% isopropanol to make the sandwich clip of the routine procedure;

[0053] 6. Put it in the electrophoresis tank, use constant pressure 30w to transfer the membrane, 1-2h, depending on the amount of sample loaded;

[0054] 7. After the transfer is comp...

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Abstract

The invention provides a transfer buffer for promoting residence of small molecular proteins on a PVDF membrane, wherein the transfer buffer uses 8-20% isopropanol as a PVDF membrane activator, simultaneous detection of small-molecule proteins and macromolecular proteins can be carried out only by using the uniform 0.45[mu]M PVDF membrane, and a new transfer solution formulation is verified by optimally searching the type and concentration of PVDF membrane activators, that is, 8-20% isopropanol used as a wet transfer buffer of 0.45[mu]M PVDF membrane can improve the resident effect of small molecular proteins, and has high repeatability, good retention effect, simple operation, broad application prospect and great market value.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a transfer solution for promoting the residence of small molecular proteins on PVDF membranes. Background technique [0002] Western blotting (immunoblotting test), or Western Blot, is an experimental method commonly used in molecular biology, biochemistry and immunogenetics, and is the gold standard for protein qualitative and quantitative detection. The main steps include: preparing SDS-PAGE gel , SDS-PAGE electrophoresis separation, transfer after electrophoresis (transfer protein molecules from PAGE to membrane for immobilization), membrane blocking, primary antibody incubation, secondary antibody incubation, ECL liquid color development and fluorescence signal acquisition and analysis, etc. The basic principle is to stain the cells or biological tissue samples treated by gel electrophoresis through specific antibodies, and obtain information about the expression of specific protei...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/533G01N33/561
CPCG01N33/533G01N33/561
Inventor 张磊王文天赵慧娟池颖李慧媛孙婷
Owner INST OF HEMATOLOGY & BLOOD DISEASES HOSPITAL CHINESE ACADEMY OF MEDICAL SCI & PEKING UNION MEDICAL COLLEGE
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