Method for extracting protein from paraffin-embedded tissue

A technology of paraffin embedding and protein, which is applied in the field of protein extraction and protein extraction from paraffin embedded tissue, achieving the effect of low cost, short time, simple and convenient operation

Inactive Publication Date: 2006-07-19
SOUTHERN MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The application of this technology is mostly based on the extraction of proteins from fresh tissues or cultured cells and then electrophoresis, and how to dewax paraffin-embedded tissues to separate high-quality proteins from them, and finally use two-dimensional electrophoresis for protein expression and further analysis and disease It is a blank in the current international research in this field

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  • Method for extracting protein from paraffin-embedded tissue

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] 1. Protein extraction from paraffin-embedded tissues:

[0033] 1. The paraffin-embedded specimens of colorectal cancer biopsied in the pathology department from 2004 to 2005 were collected, and the paraffin-embedded tissues were cut into 9 μm thick slices and placed in EP tubes;

[0034] 2. Add xylene to act at 37°C for 2 hours, centrifuge at 100rpm for 3 minutes, pour off the xylene, repeat 3 times, and observe the smoothness of the tube wall with the naked eye;

[0035] 3. Gradient hydration with 100%, 95%, 85%, and 75% ethanol, act at room temperature for 30 minutes, centrifuge at 100 rpm for 3 minutes, discard the supernatant, and finally air-dry for 5 minutes;

[0036] 4. Add the lysate (7M urea, 2M thiourea, 4% CHAPS, 65mM DTT, 1mM PMSF, 1% carrier ampholyte) according to the volume of 1:6, oscillate and mix evenly to obtain the extract, and repeat the extract in liquid nitrogen Freeze and thaw 3 times, after 60 minutes at room temperature, centrifuge at 15,000 r...

Embodiment 2

[0040] 1. Protein extraction from paraffin-embedded tissues:

[0041] 1. The paraffin-embedded specimens of colorectal cancer biopsied in the pathology department from 2004 to 2005 were collected, and the paraffin-embedded tissues were cut into 8 μm thick slices and placed on glass slides;

[0042] 2. Immerse in xylene at room temperature for 2 hours, replace xylene, repeat 3 times;

[0043] 3. Immerse in 100%, 95%, 85%, 75% ethanol for gradient hydration, act at room temperature for 30 minutes, and finally air dry for 5 minutes;

[0044]4. Add RIPA lysate (containing 1mM PMSF) according to the volume of 1:10, shake and mix well to obtain the extract, and process the extract by ultrasonic fragmentation for 6 times until the solution is clear and not viscous. The processing time is 5 seconds, intermittent The time is 10 seconds, the power is 120W, then add 50 μg / ml RNaseA and 200 μg / ml DNaseI, act at room temperature for 60 minutes, centrifuge at 15000 rpm for 30 minutes, and ...

Embodiment 3

[0047] 1. Protein extraction from paraffin-embedded tissues:

[0048] 1. The paraffin-embedded specimens of colorectal cancer biopsied in the pathology department from 2004 to 2005 were collected, and the paraffin-embedded tissues were cut into 9 μm thick slices and placed in EP tubes;

[0049] 2. Add xylene to act at 37°C for 2 hours, centrifuge at 100rpm for 3 minutes, pour off the xylene, repeat 3 times, and observe the smoothness of the tube wall with the naked eye;

[0050] 3. Gradient hydration with 100%, 95%, 85%, and 75% ethanol, act at room temperature for 30 minutes, centrifuge at 100 rpm for 3 minutes, discard the supernatant, and finally air-dry for 5 minutes;

[0051] 4. Add the lysate (7M urea, 2M thiourea, 4% CHAPS, 65mM DTT, 1mM PMSF, 1% carrier ampholyte) according to the volume of 1:5, oscillate and mix evenly to obtain the extract, and repeat the extract in liquid nitrogen Freeze and thaw 4 times, then add 100 μg / ml RNaseA and 100 μg / ml DNaseI, react at roo...

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Abstract

The extraction method for protein from paraffin embedded tissue comprises: cutting the paraffin embedded tissue; dewaxing with xylene, hydrating with ethanol; finally, adding cracking liquid and nuclease for centrifugation. This invention needs short time and low cost with high efficiency.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to protein extraction technology, in particular to a method for extracting protein from paraffin-embedded tissue. Background technique [0002] A large number of paraffin-embedded tissues accumulated in the archives of the hospital's pathology department are a reliable source of materials for molecular biology research. Goelz (1985) and Dubeau et al. (1986) successfully extracted high-quality DNA from wax blocks, which can fully meet the needs of some tumor molecular biology research, ending the history of DNA research relying on fresh or frozen tissues and cells , diagnosis and differential diagnosis research and patient prognosis assessment are of great value. Subsequently, the paraffin-embedded tissue RNA extraction and mRNA amplification technology established by Erlander (2003) was successfully applied to microarray expression profiling analysis, which fully reflected the applicat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K1/14C12P21/00
Inventor 赵亮丁彦青
Owner SOUTHERN MEDICAL UNIVERSITY
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