CN108828236A - A method of ancient times woolen knitwear relic is detected based on Western blot - Google Patents
A method of ancient times woolen knitwear relic is detected based on Western blot Download PDFInfo
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- CN108828236A CN108828236A CN201810968650.3A CN201810968650A CN108828236A CN 108828236 A CN108828236 A CN 108828236A CN 201810968650 A CN201810968650 A CN 201810968650A CN 108828236 A CN108828236 A CN 108828236A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
Abstract
The present invention relates to historical relic detection fields, disclose a kind of method based on Western blot detection ancient times woolen knitwear relic, the present invention is first prepared for the wool secondary antibody of carbon quantum dot label, modern wool and ancient times woolen knitwear relic are extracted with metal salt method and reduction method later, carry out PAGE gel electrophoresis, it is dyed with carbon quantum dot solution, observes electrophoretic image.Obtained protein band is transferred on polyvinylidene fluoride film, after the secondary antibody marked through wool primary antibody and carbon quantum dot is incubated for, immune band can be observed in gel imaging system, identifies ancient times woolen knitwear.Chemical levels are few in the present invention, react mild, environmentally friendly；When detecting to ancient times woolen knitwear, have many advantages, such as that amount of samples is few, sensitive, accurate, intuitive.
Description
Technical field
The present invention relates to historical relic detection fields more particularly to a kind of based on Western blot detection ancient times woolen knitwear relic
Method.
Background technique
Chinese history is long, and being unearthed from the grave of Chinese early stage has a large amount of wool product, and quantity still weaves it
Luxury aspect all allows people to acclaim as the acme of perfection.These unearthed woolen knitwears are studied, for understanding origin and the minority people of ancient times woolen knitwear
There is very important cultural exchanges between race's dress ornament, nationality.
In ancient times in woolen knitwear remnants, wool remnants is mainly shown as woolen knitwear relic, and this kind of remnants is imbedded in ground for a long time
It in lower environment, is influenced by environmental factors such as water, heat, oxygen, microorganisms, denaturation and aging drop can occur for wool keratin
Solution.And the aging of fiber has been further speeded up because of the acute variation of environmental condition after being unearthed, therefore, it is conceivable that, very long
In historical floods, current year be embedded to woolen knitwear of the grave perhaps in ruins lose already original pattern be degraded into peptide fragment or
Amino acid can not visually identify, and age more early evidence is more difficult to seek, and brings to the identification and Protective strategy of wool class historical relic
Very big difficulty.
Currently, common analysis and detection technology, such as FTIR spectrum, Raman spectrum, X- diffraction analysis etc. are encountering
It is often helpless when this residue analysis, it is most of also to rest on to fiber both at home and abroad for the identification of ancient times woolen knitwear
In the analysis of form, the qualification result obtained also not full accuracy；Therefore, it is next that a kind of more scientific means are found
Identification ancient times woolen knitwear is very important.
Summary of the invention
In order to solve the above-mentioned technical problems, the present invention provides one kind to detect ancient times woolen knitwear relic based on Western blot
Method.When detecting using the method for the present invention to ancient times woolen knitwear relic, has the characteristics that intuitive, accurate, high sensitivity.
The specific technical solution of the present invention is：A method of ancient times woolen knitwear relic, packet are detected based on Western blot
Include following steps：
1）The preparation of carbon quantum dot：Water and neopelex are added in pentane, oil-phase solution is made；Life is added
Substance tar aqueous solution, normal propyl alcohol and neopelex, oscillation obtain reverse micro emulsion；Lauryl amine, ultrasound, horse is added
Not kept the temperature in furnace, it is cooling, it is demulsified, carbon quantum dot solution is made in centrifugation；Then it takes half through dialysis purification, freeze-drying, obtains
Carbon quantum dot.
The present invention is using the pollutant biomass coke tar that should be removed in script industrial production as raw material, abundance, and
And solve the problems, such as pollutant process, play the role of turning waste into wealth.
Carbon quantum dot solution is prepared with reverse microemulsion process, yield is much higher than general preparation method, and carbon quantum dot grain
Degree is uniform, size adjustable.
2）The preparation of carbon quantum dot label wool keratin secondary antibody：Carbon quantum dot, carbodiimides and secondary antibody are added to
In the phosphate buffered saline solution of 9-11mmol/L, at room temperature stir 2-3 h after, with phosphate buffered saline solution to solution into
Row repeatedly washing centrifugation obtains carbon quantum dot label wool keratin secondary antibody.
Carbon quantum dot is after coupling, fluorescence capability enhancing, while can also be to avoid carbon quantum dot particle aggregation phenomenon.
3）The pH of the EWNN solution of 0.03-0.06 mol/L is adjusted to 2.5-2.8 with tartaric acid, by this solution with
350-450 μ l step 1）Remaining carbon quantum dot solution is uniformly mixed, and obtained carbon quantum polishes sol solution.
4）Ancient times woolen knitwear sample and modern wool samples are weighed respectively, is cleaned with deionized water, are dried；It is soaked respectively in pH
1-2h in the HBr solution of=1.5-2.5, is washed by rubbing with the hands with deionized water, drying；With 1：The bath raio addition of 15-25 contains 0.05-
It is submerged in the solution of the lauryl sodium sulfate of the LiBr and 0.02-0.04mol/L of 0.15mol/L, adjusting pH is 8-11, is surpassed
Sound is put into microwave reactor Irradiation Hydrolysis 6-8min.
Lauryl sodium sulfate can form huge micella with keratin, and then-the SH generated is protected to be oxidized, and be conducive to
The generation of reduction reaction prevents from precipitating, simultaneously because sodium dodecyl sulfate solution has very big surface energy, to enable wool
It is come into full contact with reducing agent, reduces the reaction time effectively.Available molecular weight height, highly concentrated keratin solution.
Metal salt LiBr has great destruction to the hydrogen bond in wool molecule, can with reducing substances collective effect
Keratin is extracted to dissolve wool.Compared to other metal salts, LiBr has better dissolution rate and recovery rate.
Ultrasound can make turbid be uniformly dispersed with assisting hydrolyzing, improve the recovery rate of albumen.Microwave can promote the molten of wool
It is swollen, make the molecular vibrations such as solvent, improve internal energy of molecular, increases hydrolysis effect, keep wool more soluble, to reduce sequential reduction
Required temperature.Because extracting the complexity of woolen system, ultrasound, the effect of microwave hydrolysis are different from extracting other albumen, cannot
It embodies, is shown in final dissolution rate, the increase of recovery rate and the reduction of sequential reduction temperature at once.And because extracting
The particularity of woolen system, ultrasonic-microwave cannot be simply added extraction step, temperature and time as extracting other albumen
It needs to adjust, by repetition test, optimal conditions are determined on the hydrolysing step before placing it in reduction.
5）It is taken out from reactor, two samples is poured into three-necked flask respectively, three-necked flask is placed on thermostat water bath
In, 70-90 DEG C of bath temperature is adjusted, leads to nitrogen 10-20 minutes, is slowly added into (the 2- carboxylic of 2.5-3.5 mol/L tri- of 10-25ml
Ethyl) phosphine solution, leads to nitrogen gas stirring, dissolves wool, be filtered by vacuum, remove not molten wool and impurity.
Three (2- carboxyethyl) phosphines are a kind of very effective thio-alcohol reducing agents.The stability of the reagent in aqueous solution and
Dissolubility is all fine.It is also good in acid, in alkaline solution stability.Compared with the thio-alcohol reducing agent of other classifications, three
(2- carboxyethyl) phosphine is more efficient disulfide bond reducing agent, and reduction effect is not easy to change because of the variation of pH.Using three
(2- carboxyethyl) phosphine excludes oxygen when restoring, and prevents its oxidation by air.
6）It is separately added into 20-35g ammonium sulfate, is stirred evenly, filtrate pH is adjusted, 8-15min is centrifuged, by lower layer's solution in 2-
With bag filter dialysis 50-60h under the conditions of 6 DEG C；Cryogenic vacuum is concentrated into the 1/4-1/2 of original volume, obtains wool keratin extraction
Liquid A, B.
With ammonium sulfate precipitation, temperature coefficient is small, and solubility is big, is not easy to cause protein denaturation.
It dialyses in refrigerator, preventing protein, desalination denaturation is precipitated at relatively high temperatures, and more stable, the rate of recovery is higher.
7) PAGE gel electrophoresis：Take wool keratin extracting solution A, B in 2* sample buffer by 1: 0.8-1.2 ratio
Example mixing, heating water bath heat 1.5-2.5 min at 93-97 DEG C, make protein denaturation；After being cooled to room temperature, sheep is taken respectively
Gel loading wells is added in each 10-20 μ l of feather keratin extracting solution, carries out PAGE gel electrophoresis；Immerse step 3）Gained carbon
Quantum polishes sol solution, and 2.5-3.5h is vibrated on shaking table with low speed, observes the electrophoretic image of sample.
Gel is polished with carbon quantum, high sensitivity, electrophoretic image is clear, high resolution.It is solidifying to solve traditional SDS-PAGE
The problem that gel electrophoresis image is smudgy.
8）Electrotransfer：Glue after electrophoresis is laid in and is turned on the processed polyvinylidene fluoride film of buffer through methanol and electricity,
It is clipped among filter paper and sponge and is assembled into transhipment interlayer, be put into wet type membrane-transferring device, constant current transferring film.
The polyvinylidene fluoride film used is first impregnated with methanol, it is therefore an objective to the group on schedule above polyvinylidene fluoride film is activated,
It is easier it in conjunction with electronegative protein.
9）Film imaging：After the completion of electrotransfer, polyvinylidene fluoride film is taken out, is put into gel imaging system and is imaged, observes egg
Informal voucher band transfer case.
Film is imaged, the transfer case of protein band is observed, facilitates and grasps experiment progress, optimizes experimental procedure.
10）Closing：Polyvinylidene fluoride film is taken out, washes film 4-6min with the triethanolamine buffered saline solution of 10-20mL, is used
Confining liquid handles 50-70min at room temperature；
11）Immune processing：With antibody diluent with 950-1050:1 volume ratio dilutes primary antibody, and film is immersed in primary antibody dilution
In, shaking table is incubated for 1 ~ 2 h under room temperature；Film is washed with TBST liquid after taking-up film, immerses the diluted carbon quantum dot of 8-12mL later
It is incubated for 1-2 h under room temperature in label wool keratin secondary antibody；
12）Shine colour developing：Directly will it is immune treated that film is imaged in gel imaging system, using carbon quantum dot in ultraviolet light
The performance of the lower transmitting fluorescence of irradiation, obtains immunoblotting image；If two samples all obtain fluorescent bands, illustrate that ancient times woolen knitwear is residual
Piece belongs to wool；If only modern wool has fluorescent bands, illustrate that ancient times woolen knitwear relic is not belonging to wool.
Preferably, step 1）In, the preparation method of carbon quantum dot is specific as follows：To 15-25 m L organic solvent positive penta
In alkane, addition molal weight ratio is 18-22:Oil-phase solution is made in 1 water and neopelex；Into oil-phase solution,
The biomass char oil solution that mass fraction is 45-55% is added, adding molal weight ratio is 1.8-2.2:1 normal propyl alcohol and
Neopelex firmly rocks, and obtains reverse micro emulsion, stands 10-13h；Into reverse micro emulsion, 0.2- is added
0.4 g lauryl amine, ultrasound 8-12 min, is poured into the reaction kettle containing polytetrafluoroethylliner liner at room temperature, in 110-130 DEG C of Muffle
2-4 h is placed in furnace, is taken out, cooled to room temperature；It is demulsified with anhydrous methanol, for several times, it is molten that carbon quantum dot is made in centrifugation for washing
Liquid；The bag filter dialysis 20-28 h that half carbon quantum dot solution molecular cut off is 1000 is taken, freeze-drying obtains carbon amounts
Sub- point.
Preferably, step 4）In, ultrasonic concrete operations are：At room temperature with the power ultrasound 30- of 190-210W
50min。
Preferably, step 5）In, logical nitrogen gas stirring is specially that nitrogen is continually fed into three-necked flask, is stirred using machinery
It mixes and 1.8-2.2 h is stirred to wool dissolution with the speed of 180-220r/min, solution becomes clarification.
Preferably, step 6）In, the filtrate pH that adjusts is specially the sulfuric acid solution regulating step 9 for using 4-5mol/ L）
Obtained filtrate pH value is to wool keratin isoelectric point 4.1-4. 3.
It saltouts and is used in combination with two methods of isoelectric precipitation, increase the recovery rate of keratin.
Preferably, the molecular cut off of the bag filter is 3500-4000.
The lesser bag filter of molecular cut off is selected, the loss of small molecular protein is reduced, greatly increases recovery rate.Because ancient
For woolen knitwear there are signs of degradation, many protein degradations are at the lesser polypeptide of molecular weight, amino acid, so reducing small molecule egg
White loss detects ancient times woolen knitwear extremely important.
Preferably, step 7）In, the sample buffer is 1.8-2.2ml 0.4-0.6mol/L tri-（Methylol）Ammonia
Methylmethane-HCl, 1.8-2.2ml glycerol, 1.8-2.2ml 18-22wt% lauryl sodium sulfate, 0.4-0.6mol 0.08-
0.12wt% bromophenol blue, the mixed solution of 0.8-1.2ml beta -mercaptoethanol；Three（Methylol）The pH of aminomethane-HCl is 6.7-
6.9。
Preferably, step 7）In, gel electrophoresis concrete operations are：Voltage is adjusted to 28-32V electrophoresis 18-22min；When
After sample is completely into separation gel, then voltage is tuned into 80-100V；When the bromophenol blue of sample is migrated to glass offset plate bottom about
When 0.8-1.2cm, electrophoresis can be stopped.
Preferably, step 8）Electrotransfer operation in, the polyvinylidene fluoride film used first with methanol impregnate 50-70s, then
Electricity consumption turns buffer immersion；Filter paper used, sponge electricity consumption turn buffer infiltration, poly- according to sponge-filter paper-running gel-
Vinylidene fluoride film-filter paper-sponge sequence assembling transhipment interlayer, constant current transferring film is 1 ~ 2 h of transferring film under 100mA constant current；This
Operation uses wet process transferring film, and membrane-transferring device need to be placed in ice bath by heat release in transfer process.
Preferably, step 10）In, after being incubated for primary antibody and secondary antibody, film is washed three times with TBST liquid on shaking table at room temperature, often
Secondary 4-6min；The confining liquid is 5wt% skimmed milk power.
Preferably, step 11）In, the antibody diluent is TBST liquid.
It is compared with the prior art, the beneficial effects of the invention are as follows：
（1）The preferred carbon quantum dot of the present invention is used as the dyeing liquor and image forming material of PAGE gel electrophoresis simultaneously, is quick on the draw,
Electrophoretic image is clear, high resolution, solves the problems, such as that traditional PAGE gel electrophoretic image is fuzzy.
（2）The present invention prepares carbon quantum dot using reverse microemulsion process, and yield is much higher than arc discharge method, hydrothermal synthesis
The general preparation methods such as method, laser ablation method, and carbon quantum dot epigranular, size adjustable.Select biomass starting material tar
For industrial waste, solve raw material and pollution two hang-ups of processing at one stroke.
（3）The present invention is combined metal salt method and reduction method extracts wool keratin, preferably to the hydrogen in wool molecule
Key has the metal salt LiBr of great destruction, with strong reducing property three (2- carboxyethyl) phosphine collective effect, compensates for metal salt
The low defect of method dissolution rate, dissolution rate ratio are applied alone metal salt method and reduction method all high.
（4）Dosage of the present invention is few, and detection keratin is very sensitive, accuracy is high, is particularly suitable for identifying of the remote past, degradation
Serious ancient times woolen knitwear relic.
（5）The present invention is hydrolyzed using ultrasound, microwave assisted, makes wool be easier to be swollen, turbid is made to be uniformly dispersed, and improves molecule
Interior energy, reduces sequential reduction required temperature, optimizes metal salt method and reduction method combines the method for extracting wool keratin.
（6）The present invention will saltout to be used in combination with two methods of isoelectric precipitation, increases the recovery rate of keratin.
Specific embodiment
The present invention will be further described with reference to the examples below.
Embodiment 1：
1）The preparation of carbon quantum dot：Into 20 m L organic solvent pentanes, it is 20 that molal weight ratio, which is added,:1 water and 12
Sodium alkyl benzene sulfonate prepares oil-phase solution；Into oil-phase solution, the biomass char oil solution that mass fraction is 50% is added, then
It is 2 that molal weight ratio, which is added,:1 normal propyl alcohol and neopelex, firmly rocks, and obtains reverse micro emulsion, stands
12h；Into reverse micro emulsion, 0.3 g lauryl amine is added, 10 min of ultrasound, are poured into containing the anti-of polytetrafluoroethylliner liner at room temperature
It answers in kettle, 3 h is placed in 120 DEG C of Muffle furnaces, take out, cooled to room temperature.Be demulsified with anhydrous methanol, washing for several times, from
Carbon quantum dot solution is made in the heart.Bag filter 24 h of dialysis for being 1000 by half solution molecular cut off, freeze-drying can
Obtain carbon quantum dot powder.
2）The preparation of carbon quantum dot label wool keratin secondary antibody：The addition of carbon quantum dot, carbodiimides and secondary antibody is pressed
1:20:20 mass ratio is added in the phosphate buffered saline solution of 10mmol/L, after stirring 2.5 h at room temperature, uses phosphoric acid
Buffer salt solution carries out repeatedly washing centrifugation to solution, and the wool keratin secondary antibody of carbon quantum dot label can be obtained.
3）The pH of the EWNN solution of 0.05 mol/L is adjusted to 2.6 with tartaric acid, by this solution and 400 μ l steps 1）
Gained carbon quantum dot solution is uniformly mixed, and dye sol solution is made.
4）Ancient times woolen knitwear sample and modern wool samples are weighed respectively, is cleaned with deionized water, are dried；It is soaked respectively in pH
1.5h in=2 HBr solution, is washed by rubbing with the hands with deionized water, drying；With 1：20 bath raio be added containing 0.1mol/L LiBr and
It is submerged in the solution of 0.03mol/L lauryl sodium sulfate, adjusts pH value to 10, at room temperature with the power ultrasound of 200W
40min is put into microwave reactor Irradiation Hydrolysis 7min.
5）It is taken out from reactor, two samples is poured into three-necked flask respectively, three-necked flask is placed on thermostat water bath
In, 80 DEG C of bath temperature are adjusted, leads to nitrogen 15 minutes, is slowly added into 3 mol/L tri- (2- carboxyethyl) phosphine solution of 20ml, lead to
Nitrogen stirs 2 h using mechanical stirring with the speed of 200r/min, dissolves wool, be filtered by vacuum, and removes not molten wool and miscellaneous
Matter.
6）It is separately added into 30g ammonium sulfate, is stirred evenly, adjusts pH value to wool angle egg with the sulfuric acid solution of 4.5mol/ L
White isoelectric point 4.1-4. 3.It is centrifuged 12min, by lower layer's solution with bag filter dialysis 55h under the conditions of 3 DEG C（Molecular cut off is
3500）；Cryogenic vacuum is concentrated into the 1/3 of original volume, obtains wool keratin extracting solution A, B.
7) PAGE gel electrophoresis：Take wool keratin extracting solution A, B in 2* sample buffer（2ml pH is 6.8
0.5mol/L tri-（Methylol）Aminomethane-HCl, 2ml glycerol, 20% lauryl sodium sulfate of 2ml, 0.1% bromine of 0.5mol
Phenol is blue, the mixed solution of 1ml beta -mercaptoethanol）It is mixed in 1: 1 ratio, heating water bath heats 2 min at 95 DEG C, makes protein
Denaturation.It after sample is cooled to room temperature, takes 15 μ l of wool keratin extracting solution A, B that gel loading wells is added respectively, carries out SDS-
PAGE gel electrophoresis；Immerse step 3）Gained carbon quantum polishes sol solution, and 3h is vibrated on shaking table with low speed, observes the electricity of sample
Swimming image.
Wherein, gel electrophoresis concrete operations are：Voltage is adjusted to 30V electrophoresis 20min.When sample is completely into separation gel
Afterwards, then by voltage it is tuned into 90V.When the bromophenol blue of sample is migrated to glass offset plate bottom about 1cm, electrophoresis can be stopped.
8）Electrotransfer：Glue after electrophoresis is laid in and is turned on the processed polyvinylidene fluoride film of buffer through methanol and electricity,
It is clipped among filter paper and sponge and is assembled into transhipment interlayer, filter paper used, sponge need electricity consumption to turn buffer infiltration, according to sea
Silk floss-filter paper-running gel-polyvinylidene fluoride film-filter paper-sponge sequence assembling transhipment interlayer, is put into wet type transferring film dress
In setting, membrane-transferring device need to be placed in ice bath by 1.5 h of transferring film under 100mA constant current, heat release in transfer process.
9）Film imaging：After the completion of electrotransfer, polyvinylidene fluoride film is taken out with tweezers, is put into gel imaging system and is imaged,
Observe protein band transfer case.
10）Closing：Polyvinylidene fluoride film is taken out, film 5min is washed with the triethanolamine buffered saline solution of 15mL, with closing
Liquid（5% skimmed milk power）1h is handled at room temperature.
11）Immune processing：Use antibody diluent（TBST liquid）With 1:1000 dilution proportion primary antibody, is immersed in primary antibody for film
In dilution, shaking table is incubated for 1.5 h under room temperature；After taking out film, film is washed three times with TBST liquid on shaking table at room temperature, every time
Five minutes, immerse 10mL later with 1:In the secondary antibody liquid of the carbon quantum dot label of 1000 dilution proportion, it is incubated under room temperature
1.5 h wash film three times with TBST liquid on shaking table at room temperature after taking out film, and five minutes every time.
12）Shine colour developing：Directly will it is immune treated that film is imaged in gel imaging system, existed using carbon quantum dot
The performance for emitting fluorescence under ultraviolet light, obtains immunoblotting image.If two samples all obtain fluorescent bands, illustrate ancient times hair
Fabric relic belongs to wool；If only modern wool has fluorescent bands, illustrate that ancient times woolen knitwear relic is not belonging to wool.
Embodiment 2：
1）The preparation of carbon quantum dot：Into 15 m L organic solvent pentanes, it is 18 that molal weight ratio, which is added,:1 water and 12
Sodium alkyl benzene sulfonate prepares oil-phase solution；Into oil-phase solution, the biomass char oil solution that mass fraction is 45% is added, then
It is 1.8 that molal weight ratio, which is added,:1 normal propyl alcohol and neopelex, firmly rocks, and obtains reverse micro emulsion, stands
10h；Into reverse micro emulsion, 0.2 g lauryl amine is added, 8 min of ultrasound, pour into the reaction containing polytetrafluoroethylliner liner at room temperature
In kettle, 2 h are placed in 110 DEG C of Muffle furnaces, are taken out, cooled to room temperature.Be demulsified with anhydrous methanol, washing for several times, from
Carbon quantum dot solution is made in the heart.The bag filter dialysis 20h for being 1000 by half solution molecular cut off, freeze-drying can obtain
To carbon quantum dot powder.
2）The preparation of carbon quantum dot label wool keratin secondary antibody：The addition of carbon quantum dot, carbodiimides and secondary antibody is pressed
1:20:20 mass ratio is added in the phosphate buffered saline solution of 9mmol/L, after stirring 2 h at room temperature, uses phosphoric acid buffer
Salting liquid carries out repeatedly washing centrifugation to solution, and the wool keratin secondary antibody of carbon quantum dot label can be obtained.
3）The pH of the EWNN solution of 0.03 mol/L is adjusted to 2.5 with tartaric acid, by this solution and 350 μ l steps 1）
Gained carbon quantum dot solution is uniformly mixed, and dye sol solution is made.
4）Ancient times woolen knitwear sample and modern wool samples are weighed respectively, is cleaned with deionized water, are dried；It is soaked respectively in pH
1h in=1.5 HBr solution, is washed by rubbing with the hands with deionized water, drying；With 1：15 bath raio be added containing 0.05mol/L LiBr and
It is submerged in the solution of 0.02mol/L lauryl sodium sulfate, adjusts pH value to 8, at room temperature with the power ultrasound 30min of 190W,
It is put into microwave reactor Irradiation Hydrolysis 6min.
5）It is taken out from reactor, two samples is poured into three-necked flask respectively, three-necked flask is placed on thermostat water bath
In, 70 DEG C of bath temperature are adjusted, leads to nitrogen 10 minutes, is slowly added into 2.5 mol/L tri- (2- carboxyethyl) phosphine solution of 10ml,
Logical nitrogen stirs 1.8 h using mechanical stirring with the speed of 180r/min, dissolves wool, be filtered by vacuum, remove not molten wool
And impurity.
6）It is separately added into 20g ammonium sulfate, is stirred evenly, adjusts pH value to wool keratin with the sulfuric acid solution of 4mol/ L
Isoelectric point 4.1-4. 3.It is centrifuged 8min, by lower layer's solution with bag filter dialysis 50h under the conditions of 2 DEG C（Molecular cut off is
3500）；Cryogenic vacuum is concentrated into the 1/4 of original volume, obtains wool keratin extracting solution A, B.
7) PAGE gel electrophoresis：Take wool keratin extracting solution A, B in 2* sample buffer（1.8ml pH is 6.7
0.4mol/L tri-（Methylol）Aminomethane-HCl, 1.8ml glycerol, 18% lauryl sodium sulfate of 1.8ml, 0.4mol
0.08% bromophenol blue, the mixed solution of 0.8ml beta -mercaptoethanol）It is mixed in 1: 0.8 ratio, heating water bath heats 1.5 at 93 DEG C
Min makes protein denaturation.After sample is cooled to room temperature, take 10 μ l of wool keratin A, B that gel loading wells is added respectively, into
Row PAGE gel electrophoresis；Immerse step 3）Gained carbon quantum polishes sol solution, and 2.5h is vibrated on shaking table with low speed, observation
The electrophoretic image of sample.
Wherein, gel electrophoresis concrete operations are：Voltage is adjusted to 28V electrophoresis 18min.When sample is completely into separation gel
Afterwards, then by voltage it is tuned into 80V.When the bromophenol blue of sample is migrated to glass offset plate bottom about 0.8cm, electrophoresis can be stopped.
8）Electrotransfer：Glue after electrophoresis is laid in and is turned on the processed polyvinylidene fluoride film of buffer through methanol and electricity,
It is clipped among filter paper and sponge and is assembled into transhipment interlayer, filter paper used, sponge need electricity consumption to turn buffer infiltration, according to sea
Silk floss-filter paper-running gel-polyvinylidene fluoride film-filter paper-sponge sequence assembling transhipment interlayer, is put into wet type transferring film dress
In setting, membrane-transferring device need to be placed in ice bath by 1 h of transferring film under 100mA constant current, heat release in transfer process.
9）Film imaging：After the completion of electrotransfer, polyvinylidene fluoride film is taken out with tweezers, is put into gel imaging system and is imaged,
Observe protein band transfer case.
10）Closing：Polyvinylidene fluoride film is taken out, film 4min is washed with the triethanolamine buffered saline solution of 10mL, with closing
Liquid（5% skimmed milk power）50min is handled at room temperature.
11）Immune processing：Use antibody diluent（TBST liquid）With 1:950 dilution proportion primary antibody, is immersed in primary antibody for film
In dilution, shaking table is incubated for 1 h under room temperature；After taking out film, film is washed three times with TBST liquid on shaking table at room temperature, every time five
Minute, immerse 8mL later with 1:In the secondary antibody liquid of the carbon quantum dot label of 1000 dilution proportion, it is incubated for 1 h under room temperature,
After taking out film, film is washed three times with TBST liquid on shaking table at room temperature, five minutes every time.
12）Shine colour developing：Directly will it is immune treated that film is imaged in gel imaging system, existed using carbon quantum dot
The performance for emitting fluorescence under ultraviolet light, obtains immunoblotting image.If two samples all obtain fluorescent bands, illustrate ancient times hair
Fabric relic belongs to wool；If only modern wool has fluorescent bands, illustrate that ancient times woolen knitwear relic is not belonging to wool.
Embodiment 3：
1）The preparation of carbon quantum dot：Into 25 m L organic solvent pentanes, it is 22 that molal weight ratio, which is added,:1 water and 12
Sodium alkyl benzene sulfonate prepares oil-phase solution；Into oil-phase solution, the biomass char oil solution that mass fraction is 55% is added, then
It is 2.2 that molal weight ratio, which is added,:1 normal propyl alcohol and neopelex, firmly rocks, and obtains reverse micro emulsion, stands
13h；Into reverse micro emulsion, 0.4 g lauryl amine is added, 12 min of ultrasound, are poured into containing the anti-of polytetrafluoroethylliner liner at room temperature
It answers in kettle, 4 h is placed in 130 DEG C of Muffle furnaces, take out, cooled to room temperature.Be demulsified with anhydrous methanol, washing for several times, from
Carbon quantum dot solution is made in the heart.The bag filter dialysis 28h for being 1000 by half solution molecular cut off, freeze-drying can obtain
To carbon quantum dot powder.
2）The preparation of carbon quantum dot label wool keratin secondary antibody：The addition of carbon quantum dot, carbodiimides and secondary antibody is pressed
1:20:20 mass ratio is added in the phosphate buffered saline solution of 11mmol/L, after stirring 3h at room temperature, uses phosphoric acid buffer
Salting liquid carries out repeatedly washing centrifugation to solution, and the wool keratin secondary antibody of carbon quantum dot label can be obtained.
3）The pH of the EWNN solution of 0.06 mol/L is adjusted to 2.5 with tartaric acid, by this solution and 450 μ l steps 1）
Gained carbon quantum dot solution is uniformly mixed, and dye sol solution is made.
4）Ancient times woolen knitwear sample and modern wool samples are weighed respectively, is cleaned with deionized water, are dried；It is soaked respectively in pH
2h in=2.5 HBr solution, is washed by rubbing with the hands with deionized water, drying；With 1：25 bath raio be added containing 0.15mol/L LiBr and
It is submerged in the solution of 0.04mol/L lauryl sodium sulfate, adjusts pH value to 11, at room temperature with the power ultrasound of 210W
50min is put into microwave reactor Irradiation Hydrolysis 8min.
5）It is taken out from reactor, two samples is poured into three-necked flask respectively, three-necked flask is placed on thermostat water bath
In, 90 DEG C of bath temperature are adjusted, leads to nitrogen 20 minutes, is slowly added into 3.5 mol/L tri- (2- carboxyethyl) phosphine solution of 25ml,
Logical nitrogen stirs 1.8 h using mechanical stirring with the speed of 180r/min, dissolves wool, be filtered by vacuum, remove not molten wool
And impurity.
6）It is separately added into 35g ammonium sulfate, is stirred evenly, adjusts pH value to wool keratin with the sulfuric acid solution of 5mol/ L
Isoelectric point 4.1-4. 3.It is centrifuged 15min, by lower layer's solution with bag filter dialysis 60h under the conditions of 6 DEG C（Molecular cut off is
4000）；Cryogenic vacuum is concentrated into the 1/2 of original volume, obtains wool keratin extracting solution A, B.
7) PAGE gel electrophoresis：Take wool keratin extracting solution A, B in 2* sample buffer（2.2ml pH is 6.9
0.6mol/L tri-（Methylol）Aminomethane-HCl, 2.2ml glycerol, 22% lauryl sodium sulfate of 2.2ml, 0.6mol
0.12% bromophenol blue, the mixed solution of 1.2ml beta -mercaptoethanol）It is mixed in 1: 1.2 ratio, heating water bath heats 2.5 at 97 DEG C
Min makes protein denaturation.After sample is cooled to room temperature, take 20 μ l of wool keratin A, B that gel loading wells is added respectively, into
Row PAGE gel electrophoresis；Immerse step 3）Gained carbon quantum polishes sol solution, and 3.5h is vibrated on shaking table with low speed, observation
The electrophoretic image of sample.
Wherein, gel electrophoresis concrete operations are：Voltage is adjusted to 32V electrophoresis 22min.When sample is completely into separation gel
Afterwards, then by voltage it is tuned into 100V.When the bromophenol blue of sample is migrated to glass offset plate bottom about 1.2cm, electrophoresis can be stopped.
8）Electrotransfer：Glue after electrophoresis is laid in and is turned on the processed polyvinylidene fluoride film of buffer through methanol and electricity,
It is clipped among filter paper and sponge and is assembled into transhipment interlayer, filter paper used, sponge need electricity consumption to turn buffer infiltration, according to sea
Silk floss-filter paper-running gel-polyvinylidene fluoride film-filter paper-sponge sequence assembling transhipment interlayer, is put into wet type transferring film dress
In setting, membrane-transferring device need to be placed in ice bath by 2 h of transferring film under 100mA constant current, heat release in transfer process.
9）Film imaging：After the completion of electrotransfer, polyvinylidene fluoride film is taken out with tweezers, is put into gel imaging system and is imaged,
Observe protein band transfer case.
10）Closing：Polyvinylidene fluoride film is taken out, film 6min is washed with the triethanolamine buffered saline solution of 20mL, with closing
Liquid（5% skimmed milk power）70min is handled at room temperature.
11）Immune processing：Use antibody diluent（TBST liquid）With 1:1050 dilution proportion primary antibody, is immersed in primary antibody for film
In dilution, shaking table is incubated for 2 h under room temperature；After taking out film, film is washed three times with TBST liquid on shaking table at room temperature, every time five
Minute, immerse 12mL later with 1:In the secondary antibody liquid of the carbon quantum dot label of 1000 dilution proportion, it is incubated for 2 under room temperature
H washes film three times with TBST liquid on shaking table at room temperature after taking out film, and five minutes every time.
12）Shine colour developing：Directly will it is immune treated that film is imaged in gel imaging system, existed using carbon quantum dot
The performance for emitting fluorescence under ultraviolet light, obtains immunoblotting image.If two samples all obtain fluorescent bands, illustrate ancient times hair
Fabric relic belongs to wool；If only modern wool has fluorescent bands, illustrate that ancient times woolen knitwear relic is not belonging to wool.
Raw materials used in the present invention, equipment is unless otherwise noted the common raw material, equipment of this field；In the present invention
Method therefor is unless otherwise noted the conventional method of this field.
The above is only presently preferred embodiments of the present invention, is not intended to limit the invention in any way, it is all according to the present invention
Technical spirit any simple modification, change and equivalent transformation to the above embodiments, still fall within the technology of the present invention side
The protection scope of case.
Claims (10)
1. a kind of method based on Western blot detection ancient times woolen knitwear relic, it is characterised in that include the following steps：
1）The preparation of carbon quantum dot：Water and neopelex are added in pentane, oil-phase solution is made；Life is added
Substance tar aqueous solution, normal propyl alcohol and neopelex, oscillation obtain reverse micro emulsion；Lauryl amine, ultrasound, horse is added
Not kept the temperature in furnace, it is cooling, it is demulsified, carbon quantum dot solution is made in centrifugation；Then it takes half through dialysis purification, freeze-drying, obtains
Carbon quantum dot；
2）The preparation of carbon quantum dot label wool keratin secondary antibody：Carbon quantum dot, carbodiimides and secondary antibody are added to 9-
In the phosphate buffered saline solution of 11mmol/L, after stirring 2-3 h at room temperature, solution is carried out with phosphate buffered saline solution
Repeatedly washing centrifugation obtains carbon quantum dot label wool keratin secondary antibody；
3）The pH of the EWNN solution of 0.03-0.06 mol/L is adjusted to 2.5-2.8 with tartaric acid, by this solution and 350-
450 μ l steps 1）Remaining carbon quantum dot solution is uniformly mixed, and obtained carbon quantum polishes sol solution；
4）Ancient times woolen knitwear sample and modern wool samples are weighed respectively, is cleaned with deionized water, are dried；Be soaked respectively pH=
1-2h in the HBr solution of 1.5-2.5, is washed by rubbing with the hands with deionized water, drying；With 1：The bath raio addition of 15-25 contains 0.05-
It is submerged in the solution of the lauryl sodium sulfate of the LiBr and 0.02-0.04mol/L of 0.15mol/L, adjusting pH is 8-11, is surpassed
Sound is put into microwave reactor Irradiation Hydrolysis 6-8min；
5）It is taken out from reactor, two samples is poured into three-necked flask respectively, three-necked flask is placed in thermostat water bath, adjusted
70-90 DEG C of water-saving bath temperature leads to nitrogen 10-20 minutes, is slowly added into the 2.5-3.5 mol/L tri- (2- carboxyethyl) of 10-25ml
Phosphine solution leads to nitrogen gas stirring, dissolves wool, is filtered by vacuum, and removes not molten wool and impurity；
6）It is separately added into 20-35g ammonium sulfate, is stirred evenly, filtrate pH is adjusted, 8-15min is centrifuged, by lower layer's solution at 2-6 DEG C
Under the conditions of with bag filter dialyse 50-60h；Cryogenic vacuum is concentrated into the 1/4-1/2 of original volume, obtain wool keratin extracting solution A,
B；
7) PAGE gel electrophoresis：Take wool keratin extracting solution A, B mixed in 1: 0.8-1.2 ratio in 2* sample buffer
It closes, heating water bath heats 1.5-2.5 min at 93-97 DEG C, makes protein denaturation；After being cooled to room temperature, wool angle is taken respectively
Gel loading wells is added in each 10-20 μ l of protein extract, carries out PAGE gel electrophoresis；Immerse step 3）Gained carbon quantum
Sol solution is polished, 2.5-3.5h is vibrated on shaking table with low speed, observes the electrophoretic image of sample；
8）Electrotransfer：Glue after electrophoresis is laid in and turns to be clipped on the processed polyvinylidene fluoride film of buffer through methanol and electricity
It is assembled into transhipment interlayer among filter paper and sponge, is put into wet type membrane-transferring device, constant current transferring film；
9）Film imaging：After the completion of electrotransfer, polyvinylidene fluoride film is taken out, is put into gel imaging system and is imaged, observes albumen one
Band transfer case；
10）Closing：Polyvinylidene fluoride film is taken out, film 4-6min is washed with the triethanolamine buffered saline solution of 10-20mL, with closing
Liquid handles 50-70min at room temperature；
11）Immune processing：With antibody diluent with 950-1050:1 volume ratio dilutes primary antibody, and film is immersed in primary antibody dilution
In, shaking table is incubated for 1 ~ 2 h under room temperature；Film is washed with TBST liquid after taking-up film, immerses the diluted carbon quantum dot of 8-12mL later
It is incubated for 1-2 h under room temperature in label wool keratin secondary antibody；
12）Shine colour developing：Directly will it is immune treated that film is imaged in gel imaging system, using carbon quantum dot in ultraviolet light
The performance of the lower transmitting fluorescence of irradiation, obtains immunoblotting image；If two samples all obtain fluorescent bands, illustrate that ancient times woolen knitwear is residual
Piece belongs to wool；If only modern wool has fluorescent bands, illustrate that ancient times woolen knitwear relic is not belonging to wool.
2. a kind of method based on Western blot detection ancient times woolen knitwear relic as described in claim 1, which is characterized in that
Step 1）In, the preparation method of carbon quantum dot is specific as follows：Into 15-25 m L organic solvent pentane, molal weight is added
Than for 18-22:Oil-phase solution is made in 1 water and neopelex；Into oil-phase solution, addition mass fraction is 45-
55% biomass char oil solution, adding molal weight ratio is 1.8-2.2:1 normal propyl alcohol and neopelex,
It firmly rocks, obtains reverse micro emulsion, stand 10-13h；Into reverse micro emulsion, 0.2-0.4 g lauryl amine is added, at room temperature
Ultrasonic 8-12 min, is poured into the reaction kettle containing polytetrafluoroethylliner liner, and 2-4 h is placed in 110-130 DEG C of Muffle furnace, is taken
Out, cooled to room temperature；It is demulsified with anhydrous methanol, for several times, carbon quantum dot solution is made in centrifugation for washing；Take half carbon quantum
The bag filter dialysis 20-28 h that point solution molecular cut off is 1000, freeze-drying obtain carbon quantum dot.
3. a kind of method based on Western blot detection ancient times woolen knitwear relic as described in claim 1, which is characterized in that
Step 4）In, ultrasonic concrete operations are：At room temperature with the power ultrasound 30-50min of 190-210W.
4. a kind of method based on Western blot detection ancient times woolen knitwear relic as described in claim 1, which is characterized in that
Step 5）In, logical nitrogen gas stirring is specially that nitrogen is continually fed into three-necked flask, using mechanical stirring with 180-220r/min
Speed stir 1.8-2.2 h to wool dissolve, solution become clarification.
5. a kind of method based on Western blot detection ancient times woolen knitwear relic as described in claim 1, which is characterized in that
Step 6）In, the filtrate pH that adjusts is specially the sulfuric acid solution regulating step 9 for using 4-5mol/ L）Obtained filtrate pH value is extremely
Wool keratin isoelectric point 4.1-4. 3；The molecular cut off of the bag filter is 3500-4000.
6. a kind of method based on Western blot detection ancient times woolen knitwear relic as described in claim 1, which is characterized in that
Step 7）In, the sample buffer is 1.8-2.2ml 0.4-0.6mol/L tri-（Methylol）Aminomethane-HCl, 1.8-
2.2ml glycerol, 1.8-2.2ml 18-22wt% lauryl sodium sulfate, 0.4-0.6mol 0.08-0.12wt% bromophenol blue, 0.8-
The mixed solution of 1.2ml beta -mercaptoethanol；Three（Methylol）The pH of aminomethane-HCl is 6.7-6.9.
7. a kind of method based on Western blot detection ancient times woolen knitwear relic as described in claim 1, which is characterized in that
Step 7）In, gel electrophoresis concrete operations are：Voltage is adjusted to 28-32V electrophoresis 18-22min；When sample is completely into separation gel
Afterwards, then by voltage it is tuned into 80-100V；When the bromophenol blue of sample is migrated to glass offset plate bottom about 0.8-1.2cm, can stop
Electrophoresis.
8. a kind of method based on Western blot detection ancient times woolen knitwear relic as described in claim 1, step 8）In, step
Rapid 8）Electrotransfer operation in, the polyvinylidene fluoride film used first with methanol impregnate 50-70s, then electricity consumption turn buffer immersion；Institute
Filter paper, sponge electricity consumption turn buffer infiltration, according to sponge-filter paper-running gel-polyvinylidene fluoride film-filter paper-
The sequence assembling transhipment interlayer of sponge, constant current transferring film is 1 ~ 2 h of transferring film under 100mA constant current；This operation uses wet process transferring film, turns
Membrane-transferring device need to be placed in ice bath by heat release during shifting.
9. a kind of method based on Western blot detection ancient times woolen knitwear relic as described in claim 1, which is characterized in that
Step 10）In, after being incubated for primary antibody and secondary antibody, film is washed three times with TBST liquid on shaking table at room temperature, each 4-6min；The closing
Liquid is 5wt% skimmed milk power.
10. a kind of method based on Western blot detection ancient times woolen knitwear relic as described in claim 1, feature exist
In step 11）In, the antibody diluent is TBST liquid.
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