CN101985461A - Method for extracting total proteins from formaldehyde-fixed tissues or formaldehyde-fixed paraffin-embedded tissues - Google Patents
Method for extracting total proteins from formaldehyde-fixed tissues or formaldehyde-fixed paraffin-embedded tissues Download PDFInfo
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Abstract
The invention provides a method for extracting total proteins from formaldehyde-fixed tissues or formaldehyde-fixed paraffin-embedded tissues, which mainly comprises the following steps of: thoroughly removing paraffin from the paraffin-embedded tissues by using a rapid paraffin dewaxing reagent; adding a protein extraction reagent into tissue slices which are washed by double-distilled water; unlinking and completely releasing almost all proteins or polypeptides which are linked with formaldehyde through incubating processes at two temperatures; and completely precipitating all the released proteins or polypeptides by using a rapid protein precipitation reagent. Tissue total protein extract prepared by the method can be used for biotechnology experimental analysis; and a rapid, safe and convenient way is provided for western blot, enzyme linked immunosorbent assay (ELISA), mass spectrometry (MS) and other immune experiments on the downstream of pathologic tissue slices, and the clinical diagnosis of disease proteomics.
Description
Technical field
The present invention relates to biological technical field, especially clinical proteomics field is a kind of from formaldehyde fixed, or rapidly and efficiently extracts the method for gross protein in formaldehyde fixed paraffin embedding (FFPE) tissue.
Background technology
Preserved a large amount of formaldehyde fixed in hospital's pathology retaining case, or formaldehyde fixed paraffin embedding (FFPE) tissue slice sample, these samples are the overwhelming majority preserved decades, even go up a century, and paraffin-embedded tissue is the important materials of pathology molecular biology and proteomics research.Because the research of the express spectra of the cell of vitro culture is the real conditions of antimer inner cell truly, study the cell of its normal cell and disease-related, express spectra difference in the cancer cells particularly, for the progress of judging cancerization, the judgement of the type of cancer, to be convenient to diagnose and treat important meaning, therefore from formaldehyde fixed, or extract DNA in formaldehyde fixed paraffin embedding (FFPE) the tissue slice sample, mRNA, MicroRNA, SnoRNA, whole protein or the like studied is an important basic work, and can suitable extraction reagent and extracting method be to be related to the nucleic acid or the protein example that are extracted reflect true quantity and convenient in the FFPE section sample, fast, carry out the important step in downstream safely.Previous people are mainly from from formaldehyde fixed, or formaldehyde fixed paraffin embedding (FFPE) tissue slice sample extraction is that (Goelz 1985 for DNA, Dubeau 1986), then Erlander (2003) etc. extracts RNA and mRNA is used for express spectra research from FFPE tissue slice sample.
For the extraction of the importance-whole protein of the express spectra research research of FFPE tissue slice sample, because protein is by formaldehyde fixed, so exist interlinkage between protein molecule inside and the protein molecule, relatively poor and the proteinic epitope of solubility that causes protein molecule, particularly the three-dimensional epi-position of protein is by havoc, during with the protein extracting in the reagent of the routine paraffin-embedded tissue after to dewaxing, it is relatively poor that whole length protein can seriously be fractured into the recovery extent of polypeptide and epitope, so less to the extraction report of full length protein in the paraffin-embedded tissue both at home and abroad.Ikeda (1998) is with an organic solvent to containing the RIPA lysate of 2%SDS, by heating, extract amyloid protein, Shan-Rong Shi (2006) is in the method for using Ikeda, when the flesh tissue sample of same-type being extracted holoprotein with same lysate, find by collection of illustrative plates than the SDS-PAGE of comparatively fresh and paraffin-embedded tissue section, the less albumen of part content is more weak even can't detect at the western of paraffin-embedded tissue blot detection signal, and 50-10Kda zone, protein band presents smears shape, illustrate that proteinic extraction efficiency is lower, whole length protein seriously ruptures.Okaka (2005) uses high temperature to take off cured, contain urea by incubation again, the solution of 2%SDS and mercaptoethanol extracts albumen, it is clean that but paraffin can not be removed fully, the albumen that extracts is also at high temperature modified by urea, the SDS of high density is to downstream process simultaneously, and particularly proteic MS (mass spectroscopy) produces serious influence.Shan-Rong Shi (2006) has used a kind of 20mMTris-HCl of containing, pH 9.0, the solution of 2%SDS is when taking off cured tissue slice protein high temperature extraction, proteinic output increases, but SDS is not easy to remove, and can the recovery and the MS analysis of epitope be produced serious influence.
Summary of the invention
The purpose of this invention is to provide a kind ofly from formaldehyde fixed, or rapidly and efficiently extract the method for holoprotein in the tissue of formaldehyde fixed paraffin embedding (Formalin-Fixed, Paraffin-Embeded Tissue FFPE).
Base program of the present invention may further comprise the steps:
1) with the tissue of formaldehyde fixed with the protein extraction agent that does not contain stain remover under two kinds of different temperature condition incubation to discharge by the protein of interlinkage;
Perhaps, with the paraffin-embedded tissue of formaldehyde fixed earlier with behind the deparaffinization reagents dewax, again with the protein extraction agent that does not contain stain remover under two kinds of different temperature condition incubation to discharge by the protein of interlinkage;
2) centrifugal and separation supernatant, supernatant separates and washing precipitation after adding protein precipitation reagent, will precipitate the tissue of the suitable agent dissolves acquisition formaldehyde fixed of employing or the holoprotein extracting solution of the paraffin-embedded tissue of formaldehyde fixed again.
Preferable, the paraffin-embedded tissue of the tissue of described formaldehyde fixed or formaldehyde fixed is tissue slice.
In the step 1, described deparaffinization reagents comprise the organic solvent that can dissolve paraffin and with the immiscible organic solvent of paraffin.
The described organic solvent that can dissolve paraffin can be selected from toluene, dimethylbenzene, D-limonene, Skellysolve A, normal hexane, nonpolarity organic solvents such as normal heptane and octane.
The immiscible organic solvent of described and paraffin can be selected from weakly polar organic solvents such as ethanol, methyl alcohol, Virahol and propyl carbinol.
Described the method for the paraffin-embedded tissue of formaldehyde fixed with the deparaffinization reagents dewax is specially: paraffin-embedded tissue was handled 3-5 minute with the organic solvent that can dissolve paraffin earlier, then add the organic solvent processing 5-60 second immiscible with paraffin again in aforementioned processing liquid, the tissue washing after will handling again also separates.
The described organic solvent that can dissolve paraffin with the immiscible volume of organic solvent ratio of paraffin be 5: 1-20: 1, preferred 5: 1-10: 1.
Deparaffinization reagents of the present invention, be different from conventional dimethylbenzene/ethanol method, thick paraffin-embedded tissue section needs twice 10 minutes the cured process of taking off to traditional dimethylbenzene/ethanol method to 10-15 μ m, use 100%, 96%, 70% ethanol aquation respectively, twice processes of 10 minutes, the time of Xu Yaoing is usually at more than 1 hour like this, and deparaffinization reagents provided by the present invention only needs short several minutes just can realize the removal fully of paraffin.
The described protein extraction agent that does not contain stain remover is by going back original reagent, and sex change reagent and damping fluid are formed.Described original reagent selected from mercapto ethanol, DTT, the TCEP etc. of going back, suitable concentration is at 5-500mM, only concentration is at 20-200mM, and described sex change reagent is selected from urea, sulphur urine, Guanidinium hydrochloride, guanidinium isothiocyanate, sodium perchlorate, sodium iodide etc., suitable concentration is at 1-9M, and only concentration is at 3-7M.Described damping fluid composition is selected from HEPES, MOPS, and TRIS, PIPES, MES, Glycine-NaOH, buffer solution systems such as citrate, suitable concentration is at 10-1000mM, and only concentration is at 20-200mM.The pH2-10 of buffer system, only concentration is at pH5-9.
Preferably, it is as follows not contain the prescription of protein extraction agent of stain remover: 20-200mMTris-HCl, pH3-9, TCEP 20-200mM, guanidinium isothiocyanate 3-7M.
Can also add Aprotinin in the described protein extraction agent that does not contain stain remover, E-64, Leupeptin, one or more proteolytic enzyme such as PMSF suppress reagent or their mixtures.
Use the protein extraction agent incubation under two kinds of different temperature condition that does not contain stain remover in the step 1, be can reversible formaldehyde to proteinic interlinkage effect, and exist interlinkage between unwinding protein matter intramolecule and the protein molecule, dissolve fully and be discharged in the protein extraction agent with the convenient egg white matter.And adopt the proteins extraction reagent that does not contain the decontamination examination, protected the conformation of the epitope of exhausted most albumen, especially cancer or oncoprotein marker.
Described under two kinds of different temperature condition the incubation method be specially: earlier at 30-120 ℃ of following incubation 10-60 minute, preferred 60-120 ℃; Then at 30-100 ℃ of following incubation 1-5 hour, preferred 30-80 ℃.Embodiment specifically enumerate for earlier 100 ℃ of following incubations 20 minutes, then 60 ℃ of following incubations 2 hours.
The protein precipitation reagent that is adopted in the step 2 can comprise trichoroacetic acid(TCA), DOC (Sodium desoxycholate), ethanol, acetone, sulfate of ammoniac, PEG8000 (polyoxyethylene glycol 8000), ether, diethyl ether etc. also can be the mixtures that above several composition is formed according to certain quality or volume ratio.
Preferably, described protein precipitation reagent is diethyl ether and alcoholic acid mixture, and diethyl ether and alcoholic acid volume ratio are 1/3-4/1; Perhaps described protein precipitation reagent is ether and alcoholic acid mixture, and ether and alcoholic acid volume ratio are 1/2-2/1.
Described protein precipitation reagent can be precipitated out almost all protein or the polypeptide that discharges fully, when removing supernatant, has reduced sugar, fat, the pollution of salt.
The suitable solubilising reagent that the present invention narrated is experiment (Western blot, ELIS, the MS according to the downstream, the 2D electrophoresis) requiring the dissolving damping fluid directly added, can be laemmli sample buffer, IEFsample buffer, 2D sample buffer, matrix damping fluid etc.; Perhaps also can be in advance with adding a spot of micrococcal nuclease, DNA is eliminated in digestion such as benzonase, and RNA is residual, reduces viscosity, adds aforementioned dissolving damping fluid again.
The standardized experimental procedure of the present invention can design as follows:
1. it is thick to get two about 10-15um, 100mm
2Paraffin-embedded tissue is cut into slices in the clean centrifuge tube of 1.5ml, adds the 1ml octane, the outstanding concussion in high speed whirlpool 30 seconds, room temperature left standstill 5 minutes, during put upside down centrifuge tube once in a while for several times.
2. add 100ul methyl alcohol, the outstanding concussion in whirlpool 10 seconds, 10000RPM is centrifugal 2 minutes again, removes the liquid of levels.
3. clean the tissue twice of dewaxing with the distilled water of 500ul, inhale as far as possible and remove liquid, keep the tissue block precipitation.
4. add 100 μ l and do not contain the protein extraction agent of stain remover, cover lid, outstanding concussion several seconds of whirlpool.
In electric heating counteract appearance or water-bath 100 ℃ the heating 20 minutes, of short duration once centrifugal, again with organizing grinding rod that tissue slice is ground.
6. 60 ℃ of heating 2 hours in electric heating counteract appearance or water-bath, during approximately took out in per 20 minutes, concussion is once.
7.4 ℃, centrifugal 15 minutes of 12000RPM carefully transfers to supernatant in the clean centrifuge tube of a new 1.5ml.
8. the precipitation reagent that adds 1ml, the outstanding concussion in whirlpool 10 seconds kept 10 minutes in 4 ℃ environment.
9.4 ℃, centrifugal 15 minutes of 12000RPM removes supernatant and keeps precipitation.
10. the precipitation reagent that adds 0.5ml, the outstanding concussion in whirlpool 10 seconds, in 4 ℃ environment, keep 10 minutes .4 ℃, centrifugal 15 minutes of 12000RPM removes supernatant, keeps precipitation. in stink cupboard, test tube is upside down on the filter paper, allow remaining evaporate clean.
11. solid precipitation is dissolved in the suitable damping fluid, and 4 ℃ again, centrifugal 5 minutes of 12000RPM collects supernatant, is used for SDS-PAGE, experiments such as Western blot.
Solid precipitation in the described step 11 can dissolve directly or indirectly in laemmlisample buffer IEF sample buffer, 2D sample buffer, matrix damping fluid etc. according to the downstream experimental requirements; Perhaps also can be in advance with adding a spot of micrococcal nuclease, DNA is eliminated in digestion such as benzonase, and RNA is residual, reduces viscosity, adds IEF sample buffer again, 2D sample buffer etc.
The precaution that need in the invention process process:
1. for improving recovery of protein efficient in the paraffin-embedded tissue, must reduce the formaldehyde fixed time in the microsection manufacture process as far as possible, thoroughly dehydration before paraffin embedding simultaneously, the time of formaldehyde fixed is long more, proteinic extraction, organic efficiency is low more.
2. take from the paraffin-embedded tissue piece as far as possible cut thin slice, or be not closed to slide glass do not pass through painted sections such as phenodin.
3. material is fresh more or the formaldehyde fixed time is short more, and recovery of protein efficient is high more.
4. contain compound excitatory in the extraction reagent, will wear emgloves during operation, avoid being infected with skin, eyes and clothes prevent to suck mouth and nose, be infected with the skin eyes after, with clear water or normal saline flushing, seek doctor's help in case of necessity immediately.
5. for the tissue samples of formaldehyde immersion, can get the tissue samples of 5-10mg at every turn, behind the centrifugal collection of distilled water washing back, directly begin operation from step 4.
6. the protein that extracts of present method is merely able to be used for experiment in vitro, can not be used in clinical, treatment and the animal body experiment etc., and consequent consequence generally assumes no responsibility.
In time holoprotein in the paraffin-embedded tissue is extracted, carry out the research of protein expression spectrum, with the convenient generation of understanding disease, particularly cancer, development, more the back situation has crucial meaning.Present method is by special fast dewaxing reagent and protein extraction agent, incubation under two kinds of different temperature condition, can will be discharged by the protein of interlinkage, through simple centrifugation step, after albumen process washing of precipitate that contains in the supernatant and the suitable agent dissolves, the correct conformation that has kept proteinic integrity and epitope to greatest extent is particularly useful for from formaldehyde fixed, or rapidly and efficiently extracts holoprotein in the paraffin-embedded histopathologic slide of formaldehyde fixed.The holoprotein extracting solution of organizing of method preparation of the present invention can be used for WesternBlot, ELISA, 2D electrophoresis, protein chip is analyzed, the MS analysis waits the Biotechnology Experiment analysis, is the quickest at present, safety, paraffin-embedded tissue whole length protein extracting mode easily, be the Western blot in histopathologic slide downstream, the experiment of exempting from service such as ELISA, MS (mass spectroscopy) and with the clinical diagnosis of disease protein matter group provide a kind of fast, safety, approach easily.
Specific implementation method
The present invention will be further described below in conjunction with several examples, should be understood that example is not to be used to limit protection scope of the present invention:
Embodiment 1
1. it is thick to get two about 10-15um, 100mm
2Paraffin embedding renal carcinoma tissue cuts into slices in the clean centrifuge tube of 1.5ml, adds the 1ml octane, the outstanding concussion in high speed whirlpool 30 seconds, room temperature left standstill 5 minutes, during put upside down centrifuge tube once in a while for several times.
2. add 100ul methyl alcohol, the outstanding concussion in whirlpool 10 seconds, 10000RPM is centrifugal 2 minutes again, removes the liquid of levels.
3. clean the tissue twice of dewaxing with the distilled water of 500ul, inhale as far as possible and remove liquid, keep the tissue block precipitation.
4. add 100 μ l and do not contain the protein extraction agent (20mM Tris-HCl, p H 9, DTT200mM, Guanidinium hydrochloride 7M) of stain remover, cover lid, outstanding concussion several seconds of whirlpool.
In electric heating counteract appearance or water-bath 100 ℃ the heating 20 minutes, of short duration once centrifugal, again with organizing grinding rod that tissue slice is ground.
6. 60 ℃ of heating 2 hours in electric heating counteract appearance or water-bath, during approximately took out in per 20 minutes, concussion is once.
7.4 ℃, centrifugal 15 minutes of 12000RPM carefully transfers to supernatant in the clean centrifuge tube of a new 1.5ml.
8. the precipitation reagent that adds 1ml, the outstanding concussion in whirlpool 10 seconds kept 10 minutes in 4 ℃ environment.
9.4 ℃, centrifugal 15 minutes of 12000RPM removes supernatant and keeps precipitation.
10. the precipitation reagent (diethyl ether and alcoholic acid mixture, diethyl ether and ethanol volume ratio are 1/3) that adds 0.5ml.The outstanding concussion in whirlpool 10 seconds, in 4 ℃ environment, keep 10 minutes .4 ℃, centrifugal 15 minutes of 12000RPM removes supernatant, keeps precipitation. in stink cupboard, test tube is upside down on the filter paper, allow remaining evaporate clean.
11. solid precipitation is dissolved among the laemmli sample buffer, 95 degree heating 5 minutes, 4 ℃ again, centrifugal 5 minutes of 12000RPM collects supernatant, is used for Western blot.
The fresh 10mg of renal carcinoma tissue according to after testing the operation of 4-11 step, is found that than Western blot immunoblotting comparatively fresh and paraffin-embedded renal carcinoma tissue the color degree of the immunoblotting of β actin does not have notable difference.
Embodiment 2
1. get the renal carcinoma tissue in the stationary liquid of being present in of 5-10mg formaldehyde fixed 12 hours.
2. with the distilled water cleansing tissue twice of 500ul, inhale as far as possible and remove liquid, keep the tissue block precipitation.
3. add 100 μ l and do not contain the protein extraction agent (20mM HEPES, p H 3, TCEP 200mM, guanidinium isothiocyanate 3M) of stain remover, cover lid, outstanding concussion several seconds of whirlpool.
In electric heating counteract appearance or water-bath 100 ℃ the heating 20 minutes, of short duration once centrifugal, again with organizing grinding rod that tissue slice is ground.
5. 60 ℃ of heating 2 hours in electric heating counteract appearance or water-bath, during approximately took out in per 20 minutes, concussion is once.
6.4 ℃, centrifugal 15 minutes of 12000RPM carefully transfers to supernatant in the clean centrifuge tube of a new 1.5ml.
7. the precipitation reagent that adds 1ml, the outstanding concussion in whirlpool 10 seconds kept 10 minutes in 4 ℃ environment.
8.4 ℃, centrifugal 15 minutes of 12000RPM removes supernatant and keeps precipitation.
9. the precipitation reagent (ether and alcoholic acid mixture, ether and alcoholic acid volume ratio are 2/1) that adds 0.5ml.The outstanding concussion in whirlpool 10 seconds, in 4 ℃ environment, keep 10 minutes .4 ℃, centrifugal 15 minutes of 12000RPM removes supernatant, keeps precipitation. in stink cupboard, test tube is upside down on the filter paper, allow remaining evaporate clean.
10. solid precipitation is dissolved among the laemmli sample buffer, 95 degree heating 5 minutes, 4 ℃ again, centrifugal 5 minutes of 12000RPM collects supernatant, is used for the SDS-PAGE experiment.
With the fresh 10mg of renal carcinoma tissue according to the operation of experiment 3-10 step after, find than SDS-PAGE electrophoresis comparatively fresh and paraffin embedding renal carcinoma tissue, except the protein band of the interval paraffin embedding of 10-50Kda renal carcinoma tissue was smeared a little, other did not have significant difference.
Embodiment 3
1. it is thick to get two about 1015um, 100mm
2Paraffin embedding renal carcinoma tissue cuts into slices in the clean centrifuge tube of 1.5ml, adds the 1ml Skellysolve A, the outstanding concussion in high speed whirlpool 30 seconds, room temperature left standstill 5 minutes, during put upside down centrifuge tube once in a while for several times.
2. add 100ul ethylene glycol, the outstanding concussion in whirlpool 10 seconds, 10000RPM is centrifugal 2 minutes again, removes the liquid of levels.
3. clean the tissue twice of dewaxing with the distilled water of 500ul, inhale as far as possible and remove liquid, keep the tissue block precipitation.
4. add 100 μ l and do not contain protein extraction agent (20mM Mops, p H 10, mercaptoethanol 20mM, the urea 9M) cover lid of stain remover, the outstanding concussion several seconds of whirlpool.
In electric heating counteract appearance or water-bath 100 ℃ the heating 20 minutes, of short duration once centrifugal, again with organizing grinding rod that tissue slice is ground.
6. 60 ℃ of heating 2 hours in electric heating counteract appearance or water-bath, during approximately took out in per 20 minutes, concussion is once.
7.4 ℃, centrifugal 15 minutes of 12000RPM carefully transfers to supernatant in the clean centrifuge tube of a new 1.5ml.
8. the precipitation reagent that adds 1ml, the outstanding concussion in whirlpool 10 seconds kept 10 minutes in 4 ℃ environment.
9.4 ℃, centrifugal 15 minutes of 12000RPM removes supernatant and keeps precipitation.
10. the precipitation reagent (diethyl ether and alcoholic acid mixture, diethyl ether and alcoholic acid volume ratio are 4/1) that adds 0.5ml.The outstanding concussion in whirlpool 10 seconds, in 4 ℃ environment, keep 10 minutes .4 ℃, centrifugal 15 minutes of 12000RPM removes supernatant, keeps precipitation. in stink cupboard, test tube is upside down on the filter paper, allow remaining evaporate clean.
Digest 11. solid precipitation is dissolved in the benzonase damping fluid of 100ul final concentration 1u/ul, use the precipitation reagent post precipitation again, be dissolved in again among the 2D sample buffer, carry out the 2D electrophoresis.
With the fresh 10mg of renal carcinoma tissue according to the operation of experiment 4-11 step after, find that than 2D electrophoresis comparatively fresh and paraffin embedding renal carcinoma tissue except the peculiar band of benzonase, other protein difference of counting is not remarkable.
Embodiment 4
1. it is thick to get two about 10-15um, 100mm
2Paraffin embedding renal carcinoma tissue cuts into slices in the clean centrifuge tube of 1.5ml, adds the 1ml octane, the outstanding concussion in high speed whirlpool 30 seconds, room temperature left standstill 5 minutes, during put upside down centrifuge tube once in a while for several times.
2. add 100ul ethylene glycol, the outstanding concussion in whirlpool 10 seconds, 10000RPM is centrifugal 2 minutes again, removes the liquid of levels.
3. clean the tissue twice of dewaxing with the distilled water of 500ul, inhale as far as possible and remove liquid, keep the tissue block precipitation.
4. add 100 μ l and do not contain protein extraction agent (20mMTris-HCl, p H9, TCEP20mM, the Guanidinium hydrochloride 7M) cover lid of stain remover, the outstanding concussion several seconds of whirlpool.
In electric heating counteract appearance or water-bath 100 ℃ the heating 20 minutes, of short duration once centrifugal, again with organizing grinding rod that tissue slice is ground.
6. 60 ℃ of heating 2 hours in electric heating counteract appearance or water-bath, during approximately took out in per 20 minutes, concussion is once.
7.4 ℃, centrifugal 15 minutes of 12000RPM carefully transfers to supernatant in the clean centrifuge tube of a new 1.5ml.
8. the precipitation reagent that adds 1ml, the outstanding concussion in whirlpool 10 seconds kept 10 minutes in 4 ℃ environment.
9.4 ℃, centrifugal 15 minutes of 12000RPM removes supernatant and keeps precipitation.
10. the precipitation reagent (ether and alcoholic acid mixture, ether and alcoholic acid volume ratio are 1/2) that adds 0.5ml.The outstanding concussion in whirlpool 10 seconds, in 4 ℃ environment, keep 10 minutes .4 ℃, centrifugal 15 minutes of 12000RPM removes supernatant, keeps precipitation. in stink cupboard, test tube is upside down on the filter paper, allow remaining evaporate clean.
Digest 11. solid precipitation is dissolved in the benzonase damping fluid of 100ul final concentration 1u/ul, use the precipitation reagent post precipitation again, be dissolved in again among the 2D sample buffer, carry out the 2D electrophoresis, then the protein spots with β-actin and Cytokeratin takes out, and analyzes through being used for being MS after suitably handling.
The fresh 10mg of renal carcinoma tissue according to after testing the operation of 4-11 step, is found that than MS comparatively fresh and paraffin embedding renal carcinoma tissue the MS of β-actin and Cytokeratin does not have evident difference.
Claims (15)
1. method of extracting gross protein from formaldehyde fixed tissue or formaldehyde fixed paraffin-embedded tissue may further comprise the steps:
1) with the tissue of formaldehyde fixed with the protein extraction agent that does not contain stain remover under two kinds of different temperature condition incubation to discharge by the protein of interlinkage;
Perhaps, with the paraffin-embedded tissue of formaldehyde fixed earlier with behind the deparaffinization reagents dewax, again with the protein extraction agent that does not contain stain remover under two kinds of different temperature condition incubation to discharge by the protein of interlinkage;
2) centrifugal and separation supernatant separates and washing precipitation behind the supernatant adding protein precipitation reagent, will precipitate and adopt suitable solubilising reagent to dissolve the tissue of acquisition formaldehyde fixed or the gross protein extracting solution of the paraffin-embedded tissue of formaldehyde fixed.
2. the method for claim 1 is characterized in that, the paraffin-embedded tissue of the tissue of described formaldehyde fixed or formaldehyde fixed is tissue slice.
3. the method for claim 1 is characterized in that, in the step 1, described deparaffinization reagents comprise the organic solvent that can dissolve paraffin and with the immiscible organic solvent of paraffin.
4. the method for claim 1, it is characterized in that, in the step 1, with the paraffin-embedded tissue of formaldehyde fixed be: paraffin-embedded tissue was handled 3-5 minute with the organic solvent that can dissolve paraffin earlier with the method for deparaffinization reagents dewax, then add the organic solvent processing 5-60 second immiscible with paraffin again in aforementioned processing liquid, the tissue washing after will handling again also separates.
5. method as claimed in claim 4 is characterized in that, the described organic solvent that can dissolve paraffin with the immiscible volume of organic solvent ratio of paraffin be 5: 1-20: 1.
6. as the arbitrary described method of claim 3-5, it is characterized in that the described organic solvent that can dissolve paraffin is selected from toluene, dimethylbenzene, D-limonene, Skellysolve A, normal hexane, normal heptane and octane; The immiscible organic solvent of described and paraffin is selected from ethanol, methyl alcohol, Virahol and propyl carbinol.
7. the method for claim 1 is characterized in that, in the step 1, the described protein extraction agent that does not contain stain remover is formed by going back original reagent, sex change reagent and damping fluid.
8. method as claimed in claim 7 is characterized in that, the described original reagent selected from mercapto ethanol of going back, and DTT and TCEP, the concentration in the protein extraction agent is 5-500mM; Described sex change reagent is selected from urea, sulphur urine, and Guanidinium hydrochloride, guanidinium isothiocyanate, sodium perchlorate and sodium iodide, the concentration in the protein extraction agent is 1-9M; Described damping fluid is selected from HEPES, MOPS, and TRIS, PIPES, MES, Glycine NaOH and Citrate buffer solution system, the concentration in the protein extraction agent is 10-1000mM, the pH of buffer system is 2-10.
9. method as claimed in claim 8 is characterized in that, the described concentration of original reagent in the protein extraction agent of going back is 20-200mM; The concentration of described sex change reagent in the protein extraction agent is 3-7M; Described damping fluid is the buffer system of 20-200mM pH 5-9.
10. method as claimed in claim 7 is characterized in that, the described protein extraction agent that does not contain stain remover contains following component:
The 20-200mM Tris-HCl of pH3-9;
TCEP?20-200mM；
Guanidinium isothiocyanate 3-7M.
11. the method for claim 1 is characterized in that, in the step 1, the method for incubation is under two kinds of different temperature condition: earlier at 30-120 ℃ of following incubation 10-60 minute; Then at 30-100 ℃ of following incubation 1-5 hour.
12. method as claimed in claim 11 is characterized in that, the method for incubation is under two kinds of different temperature condition: earlier at 60-120 ℃ of following incubation 20-60 minute; Then at 30-80 ℃ of following incubation 1-5 hour.
13. the method for claim 1 is characterized in that, in the step 2, described protein precipitation reagent is selected from trichoroacetic acid(TCA), DOC, ethanol, acetone, sulfate of ammoniac, PEG8000, the mixing of one or more in ether and the diethyl ether.
14. method as claimed in claim 13 is characterized in that, described protein precipitation reagent is diethyl ether and alcoholic acid mixture, and diethyl ether and alcoholic acid volume ratio are 1/3-4/1; Perhaps described protein precipitation reagent is ether and alcoholic acid mixture, and ether and alcoholic acid volume ratio are 1/2-2/1.
15. the method for claim 1 is characterized in that, in the step 2, described suitable solubilising reagent is selected from laemmli sample buffer, IEF sample buffer, 2D sample buffer or matrix damping fluid.
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| CN107522771A (en) * | 2017-10-19 | 2017-12-29 | 宁夏大学 | Tapetal cell total protein extraction method |
| CN109371107A (en) * | 2018-12-25 | 2019-02-22 | 山东博思源生物技术有限公司 | A kind of rapid automatized extracting method and reagent of paraffin section tissue nucleic acid |
| CN110501437A (en) * | 2019-08-28 | 2019-11-26 | 武汉金开瑞生物工程有限公司 | The extraction of protein and determination method in a kind of paraffin embedding sample |
| CN110864939A (en) * | 2018-08-27 | 2020-03-06 | 深圳大学 | Protein extraction method and application |
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| CN110501437A (en) * | 2019-08-28 | 2019-11-26 | 武汉金开瑞生物工程有限公司 | The extraction of protein and determination method in a kind of paraffin embedding sample |
| CN110501437B (en) * | 2019-08-28 | 2021-03-23 | 武汉金开瑞生物工程有限公司 | Extraction and detection analysis method for protein in paraffin-embedded sample |
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