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Transmembrane method of electrophoretic-mobility-shift-assay (EMSA) experiment

A gel electrophoresis and membrane transfer technology, applied in the field of protein detection, can solve the problems of low overall efficiency, large consumption of reagents, and long time consumption

Inactive Publication Date: 2019-01-18
KUNMING MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The commonly used electrotransfer method is wet transfer, the voltage is between 100v-120v, and the transfer time is 30min-60min, which takes a long time, consumes a lot of reagents, and the overall efficiency is low

Method used

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  • Transmembrane method of electrophoretic-mobility-shift-assay (EMSA) experiment
  • Transmembrane method of electrophoretic-mobility-shift-assay (EMSA) experiment
  • Transmembrane method of electrophoretic-mobility-shift-assay (EMSA) experiment

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Embodiment 1

[0031] The embodiment of the present invention discloses a membrane transfer method for gel electrophoresis migration experiment, that is, according to the size of the gel, the filter paper and the nylon membrane are cut into the same size shape as the gel; according to the formula of filter paper-nylon membrane-gel-filter paper Sequentially, place the experimental materials from bottom to top to prepare a transfer film block; wherein first three layers of the filter paper are laid on the bottom, and the air bubbles are removed; then the nylon membrane paper is spread on the filter paper, and the air bubbles are removed; The gel is covered on the nylon membrane, and air bubbles are removed; finally, three layers of the filter paper are covered on the gel. Prepare a transfer solution of 0.5×TBE, refrigerate the prepared transfer solution at 4°C, absorb 3ml of the transfer solution and spray it evenly on the transfer block, and then put the transfer block into a semi-dry transfer...

Embodiment 2

[0033] The embodiment of the present invention discloses a membrane transfer method for gel electrophoresis migration experiment, that is, according to the size of the gel, the filter paper and the nylon membrane are cut into the same size shape as the gel; according to the formula of filter paper-nylon membrane-gel-filter paper Sequentially, place the experimental materials from bottom to top to prepare a transfer film block; wherein first three layers of the filter paper are laid on the bottom, and the air bubbles are removed; then the nylon membrane paper is spread on the filter paper, and the air bubbles are removed; The gel is covered on the nylon membrane, and air bubbles are removed; finally, three layers of the filter paper are covered on the gel. Prepare a transfer solution of 0.5×TBE, refrigerate the prepared transfer solution at 4°C, absorb 1ml of the transfer solution and spray it evenly on the transfer block, and then put the transfer block into a semi-dry transfer...

Embodiment 3

[0035] The embodiment of the present invention discloses a membrane transfer method for gel electrophoresis migration experiment, that is, according to the size of the gel, the filter paper and the nylon membrane are cut into the same size shape as the gel; according to the formula of filter paper-nylon membrane-gel-filter paper Sequentially, place the experimental materials from bottom to top to prepare a transfer film block; wherein first three layers of the filter paper are laid on the bottom, and the air bubbles are removed; then the nylon membrane paper is spread on the filter paper, and the air bubbles are removed; The gel is covered on the nylon membrane, and air bubbles are removed; finally, three layers of the filter paper are covered on the gel. Prepare a transfer solution of 0.5×TBE, refrigerate the prepared transfer solution at 4°C, absorb 5ml of the transfer solution and spray it evenly on the transfer block, and then put the transfer block into a semi-dry transfer...

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Abstract

The invention discloses a transmembrane method of electrophoretic-mobility-shift-assay (EMSA) experiment. In the method, a filter paper and a nylon membrane are cut to a gel size according to the gelsize; a test material is placed from a bottom to a top according to an order of filter paper-nylon membrane-gel-filter paper, and a transfer block is prepared; and 1-5 ml of freshly prepared transmembrane liquid after 4-degree cold storage are evenly sprayed on the transfer block, and then the transfer block is placed in a semi-dry transmembrane instrument for transmembrane completion. The transmembrane method of the electrophoretic-mobility-shift-assay experiment provided by the invention overcomes the technical defects that according to traditional wet transmembrane manners, consumed time islonger, reagent consumption quantity is large, and overall efficiency is low, achieves goals that a transmembrane process can be completed in 15 min, and transmembrane efficiency is improved, and hasan optimization effect on the EMSA experiment.

Description

technical field [0001] The invention relates to the technical field of protein detection, in particular to a membrane transfer method for gel electrophoresis migration experiments. Background technique [0002] Different components are distinguished by electrophoresis and transferred to a solid support. Specific reagents (antibodies) are used as probes to detect target substances. Western blotting of proteins combines the high resolution of gel electrophoresis and solid phase Immunoassays detect target substances, and Western blotting of proteins combines the high resolution of gel electrophoresis with solid-phase immunoassays. [0003] Among them, under the action of electric current, the protein is transferred from the gel to the solid phase carrier (membrane), which is the core link of the Western blotting technology of the protein. However, in the traditional gel electrophoretic migration assay (EMSA), it is necessary to transfer the protein The proteins and probes were...

Claims

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Application Information

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IPC IPC(8): G01N27/447
CPCG01N27/447
Inventor 宝福凯柳爱华戴熙廷白若兰计震华简苗苗丁喆毕云凤王峰
Owner KUNMING MEDICAL UNIVERSITY
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