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Protein extraction method and application

A protein and the resulting technology, applied in the biological field, can solve the problems of less total protein extraction, toxic to human health, and difficulty in meeting demand, and achieve stable dewaxing effect, not easy to be damaged and broken, simple and time-saving dewaxing steps Effect

Inactive Publication Date: 2020-03-06
SHENZHEN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although this method extracts proteins from paraffin-embedded tissues, the reagents used are toxic to human health, and the total amount of protein extracted is small, which is difficult to meet the needs of various proteomic methods for trace samples, so it has not been widely used.

Method used

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  • Protein extraction method and application
  • Protein extraction method and application
  • Protein extraction method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0074] A method for extracting protein from paraffin-embedded tissue, specifically comprising the steps of:

[0075] (1) Remove excess paraffin from the paraffin-embedded tissue block, cut it into tissue sections with a thickness of 28 μm, and then place it in a nested screen chamber (such as figure 2 shown);

[0076] (2) immerse the section described in step (1) into the TO transparent agent for dewaxing, and perform dewaxing treatment 3 times, soaking for 10 minutes each time;

[0077] (3) Ethanol gradient hydration treatment was performed on the tissue slices obtained in step (2). Firstly, the tissue slices were hydrated twice by immersing in absolute ethanol for 5 minutes each time, then immersed in 95% ethanol for 5 minutes, and then immersed in 75% ethanol for 5 minutes. Finally, immerse in distilled water twice, each time for 5 minutes;

[0078] (4) Drain the tissue slices obtained in step (3), collect them in a weighed 1.5mL EP tube, wash the tissue slices twice wit...

Embodiment 2

[0083] (1) Remove excess paraffin from the paraffin-embedded tissue block, cut it into tissue sections with a thickness of 20 μm, and then place it in a nested screen chamber;

[0084] (2) immerse the tissue section described in step (1) into TO transparent agent for dewaxing, and perform dewaxing treatment 4 times, soaking for 12 minutes each time;

[0085] (3) Carry out ethanol gradient hydration treatment to the tissue slices obtained in step (2), first immerse the tissue slices in absolute ethanol for hydration 3 times, each time for 5 min, then immerse in 95% ethanol for 5 min, then immerse in 75% ethanol for 5 min, Finally, immerse in distilled water 3 times, each time for 4 minutes;

[0086] (4) Drain the tissue slices obtained in step (3), collect them in a weighed 1.5mL EP tube, wash the tissue slices twice with PBS, each time for 8 min, centrifuge at 14000 rpm for 5 min, discard the supernatant, and Calculation of the weight of the slice by the method of measurement...

Embodiment 3

[0091](1) Remove excess paraffin from the paraffin-embedded tissue block, cut it into tissue sections with a thickness of 10 μm, and then place it in a nested screen chamber;

[0092] (2) immerse the tissue section described in step (1) into TO transparent agent for dewaxing, dewaxing treatment 2 times, soaking for 15min each time;

[0093] (3) Ethanol gradient hydration treatment was performed on the tissue slices obtained in step (2). Firstly, the tissue slices were hydrated once by immersing in absolute ethanol for 8 min each time, then immersed in 95% ethanol for 3 min, and then immersed in 75% ethanol for 5 min. Finally, immerse in distilled water 3 times, each time for 3 minutes;

[0094] (4) Drain the tissue slices obtained in step (3), collect them in a weighed 1.5mL EP tube, wash the tissue slices once with PBS, 10 min each time, centrifuge at 13,000 rpm for 5 min, discard the supernatant, and Calculation of the weight of the slice by the method of measurement;

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Abstract

The invention provides a protein extraction method and an application, and particularly relates to a method for extracting protein from paraffin-embedded tissues and an application. According to the method, toxic xylene is replaced by a non-toxic TO transparent agent, so that the method is safe and environment-friendly; the protein is subjected to extraction treatment by using a high-temperature boiling method, so that low-abundance protein degradation and loss caused by tissue freeze-thaw cracking or ultrasonic crushing of a conventional operation is avoided; an antigen repairing step of an antigen repairing liquid adopting microwave heating is replaced by using metal bath heating; and by virtue of match of a nest screen cloth cell, the operation is simple and feasible, and the integrityof a tissue slice can be better guaranteed. The method not only improves the extraction amount of the total protein of the paraffin tissues, but also increases the detection sensitivity to the low-abundance protein; and the overall operation process is simple, feasible and time-saving, and is safe, non-toxic and low in cost.

Description

technical field [0001] The invention belongs to the field of biotechnology, relates to a method and application of protein extraction, relates to a method and application of extracting protein from paraffin-embedded tissue, and specifically relates to a qualitative and semi-quantitative determination of paraffin-embedded tissue fixed by formalin. Methods and applications of archived total protein levels in human or animal tissue blocks. Background technique [0002] At present, most of the lesion tissue specimens resected by clinical surgery are collected by the pathology departments of hospitals at all levels. They are first fixed in paraformaldehyde, embedded in paraffin, and then sectioned, followed by HE and immunohistochemical staining, and observed and analyzed under a microscope. , and the remaining tissue samples can be archived for permanent preservation. [0003] At present, the clinical basic research and functional verification of disease proteomics mostly rely ...

Claims

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Application Information

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IPC IPC(8): G01N1/28G01N1/44
CPCG01N1/28G01N1/44
Inventor 王筠钟姗盛司潼谭辉彪
Owner SHENZHEN UNIV
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