CN110412297A - A method of sample fidelity is simulated based on Western blot silk fabric cultural relics - Google Patents
A method of sample fidelity is simulated based on Western blot silk fabric cultural relics Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 48
- 239000004744 fabric Substances 0.000 title claims abstract description 19
- 238000001262 western blot Methods 0.000 title claims abstract description 10
- 230000032683 aging Effects 0.000 claims abstract description 47
- 239000003513 alkali Substances 0.000 claims abstract description 42
- 238000004088 simulation Methods 0.000 claims abstract description 10
- 108010022355 Fibroins Proteins 0.000 claims description 39
- 239000007788 liquid Substances 0.000 claims description 35
- 239000011521 glass Substances 0.000 claims description 34
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 28
- 239000003292 glue Substances 0.000 claims description 25
- 238000001962 electrophoresis Methods 0.000 claims description 24
- 238000012546 transfer Methods 0.000 claims description 21
- 230000008569 process Effects 0.000 claims description 19
- 239000012153 distilled water Substances 0.000 claims description 18
- 102000004169 proteins and genes Human genes 0.000 claims description 17
- 108090000623 proteins and genes Proteins 0.000 claims description 17
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 15
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 15
- 239000006180 TBST buffer Substances 0.000 claims description 15
- 230000009182 swimming Effects 0.000 claims description 15
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 14
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 claims description 14
- 239000012528 membrane Substances 0.000 claims description 14
- 229920002981 polyvinylidene fluoride Polymers 0.000 claims description 14
- 238000003384 imaging method Methods 0.000 claims description 13
- 239000000243 solution Substances 0.000 claims description 13
- 240000000249 Morus alba Species 0.000 claims description 11
- 235000008708 Morus alba Nutrition 0.000 claims description 11
- 239000000427 antigen Substances 0.000 claims description 11
- 102000036639 antigens Human genes 0.000 claims description 11
- 108091007433 antigens Proteins 0.000 claims description 11
- 238000004140 cleaning Methods 0.000 claims description 10
- 239000000843 powder Substances 0.000 claims description 10
- 108010033276 Peptide Fragments Proteins 0.000 claims description 8
- 102000007079 Peptide Fragments Human genes 0.000 claims description 8
- 238000000502 dialysis Methods 0.000 claims description 8
- 238000000926 separation method Methods 0.000 claims description 8
- 238000012360 testing method Methods 0.000 claims description 8
- 239000003550 marker Substances 0.000 claims description 7
- 229910000029 sodium carbonate Inorganic materials 0.000 claims description 7
- 239000006228 supernatant Substances 0.000 claims description 7
- UDSAIICHUKSCKT-UHFFFAOYSA-N bromophenol blue Chemical compound C1=C(Br)C(O)=C(Br)C=C1C1(C=2C=C(Br)C(O)=C(Br)C=2)C2=CC=CC=C2S(=O)(=O)O1 UDSAIICHUKSCKT-UHFFFAOYSA-N 0.000 claims description 5
- 230000003139 buffering effect Effects 0.000 claims description 5
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- 230000000873 masking effect Effects 0.000 claims description 5
- 239000013078 crystal Substances 0.000 claims description 4
- 238000010438 heat treatment Methods 0.000 claims description 4
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- 230000014759 maintenance of location Effects 0.000 claims description 3
- 102000014914 Carrier Proteins Human genes 0.000 claims description 2
- 108010078791 Carrier Proteins Proteins 0.000 claims description 2
- 241001465754 Metazoa Species 0.000 claims description 2
- 238000011534 incubation Methods 0.000 claims description 2
- 238000002156 mixing Methods 0.000 claims description 2
- 239000007787 solid Substances 0.000 claims description 2
- 102000002322 Egg Proteins Human genes 0.000 claims 1
- 108010000912 Egg Proteins Proteins 0.000 claims 1
- 201000010099 disease Diseases 0.000 claims 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims 1
- 238000001514 detection method Methods 0.000 abstract description 17
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- BDERNNFJNOPAEC-UHFFFAOYSA-N propan-1-ol Chemical compound CCCO BDERNNFJNOPAEC-UHFFFAOYSA-N 0.000 description 6
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- 241000208340 Araliaceae Species 0.000 description 1
- 235000005035 Panax pseudoginseng ssp. pseudoginseng Nutrition 0.000 description 1
- 235000003140 Panax quinquefolius Nutrition 0.000 description 1
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Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/416—Systems
- G01N27/447—Systems using electrophoresis
-
- 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 technique fields, disclose a kind of method based on Western blot silk fabric cultural relics simulation sample fidelity, the present invention first prepares different silk fabric cultural relics simulation samples, influence due to different alkali ageing times to silk is on the knees of the gods, therefore the present invention is carrying out immunology detection (immunoblotting) to each group silk fabric cultural relics simulation sample, the influence of influence and immunology detection of the alkali aging to silk structure is probed into, and finishing screen selects the highest sample of fidelity.
Description
Technical field
The present invention relates to historical relic detection technique fields, more particularly to one kind to be based on Western blot silk fabric cultural relics mould
The method of quasi- sample fidelity.
Background technique
China is the country for possessing history culture of long standing and well established, and wherein silk can be described as wherein most having symbol
One of the culture logo of property.The manufacture of silk and usage history are closely connected with this Chinese thousands of years development, and silk is studied
Silk fabric historical relic can have one to be better understood by this Chinese thousands of years culture and economic development.But silk relics because
Its fragile characteristic, it tends to be difficult to which intact preserves, and it is all from Gu that the ancient silk due to obtaining at present is most of
It is excavated out in tomb, complex environment can cause the destruction for being difficult to restore to silk goods therein in tomb.In general, big portion
Sub-wire silk fabric all has occurred aging and even degrades.The microscratch overwhelming majority of silk is all present in soil, and archaeology personnel often can only
A small amount of pedotheque is obtained, and available fibroin albumen content is even more only enough to support one in this small amount of pedotheque
The requirement tested twice is unable to satisfy the demand more tested.It therefore is all that silk text is made by artificial ageing at present
Object simulates sample, is studied with silk relics simulation sample to substitute true historical relic.But how to determine silk relics simulation sample
Fidelity is a problem.
Summary of the invention
In order to solve the above-mentioned technical problems, the present invention provides one kind to be simulated based on Western blot silk fabric cultural relics
The method of sample fidelity.The present invention first prepares different silk fabric cultural relics simulation samples, since different alkali ageing times is to silk
Influence it is on the knees of the gods, therefore the present invention to each group silk fabric cultural relics simulation sample carry out immunology detection (Western blotting
Method), the influence of influence and immunology detection of the alkali aging to silk structure is probed into, and finishing screen selects the highest sample of fidelity
Product.
The specific technical proposal of the invention is: a kind of simulate sample fidelity based on Western blot silk fabric cultural relics
Method includes the following steps:
1) mulberry silk is immersed in sodium carbonate degumming liquid, heats and be stirred continuously 20-40min, it is multiple to repeat this operation；
2) after silk gum is sufficiently sloughed, insoluble fibroin distilled water flushing is multiple, it is subsequently placed at 50-70 DEG C and does
It is dry, gained fibroin is then divided into multiple groups, is added respectively into sodium hydroxide solution by bath raio 1: 90-110g/mL at 35-40 DEG C
Lower alkali aging different time；
3) it is filtered after alkali aging, gained fibroin is placed in drying, every group of fibroin at 35-40 DEG C and presses bath raio 1 respectively:
40-60g/mL is added into protein extract, the heating stirring 1.5-2.5h in 95-100 DEG C of water-bath, then will be dissolved with fibroin
The protein extract of albumen is dialysed；
4) protein extract obtained by step 3) is filtered, freeze-drying pulverizes after 1-3 days, obtains silk fibroin powder
End；
5) gum-making rack wash with distilled water takes out after dry 10-20min at 55-65 DEG C, after putting up gum-making rack, checks
Whether leak carries out subsequent glue operation, obtained tundish accompanies two pieces of glass plates of several swimming lanes as water-tight；
6) each group silk fibroin powder is dissolved separately in the fibroin albumen mixing for being made that concentration is 8-12mg/mL in CB liquid
Then liquid respectively takes the reduced form 5XSDS sample-loading buffer of 2.5-3.5ul in test tube, is then respectively adding each group fibroin albumen
Mixed liquor heats 8-12min, centrifugal treating in 95-100 DEG C of water-bath；
7) it takes supernatant to carry out electrophoretic procedures, is added in first swimming lane and does not contaminate marker for molecular weight marker, so
After sequentially add each group supernatant and carry out electrophoresis, by the glue removing between glass plate and wash with distilled water 3 to 4 after the completion of electrophoresis
It is secondary, electrophoresis result is observed by chemiluminescence imaging instrument after cleaning；
8) after observing, cut a pvdf membrane, be soaked in methanol 0.5-1.5min and excited, then by pvdf membrane and
Sponge filter paper is dipped in transfering buffering liquid, is carried out building for sandwich structure, is then transferred, ice bag is used in transfer process
It is wrapped in reaction unit surrounding, Transfer current 190-210mA, transfer process continues 20-40min；
9) 18-22mL confining liquid is poured on pvdf membrane, is outwelled after shaking table closing 0.5-1.5h, 8-12mL TBST is added
The primary antibody diluted, primary antibody shaking table are incubated for 0.5-1.5h, are then cleaned repeatedly with TBST, each dosage 18-22mL, shaking table 8-
12min after cleaning repeatedly, adds the secondary antibody after 8-12mL dilutes and is incubated for 0.5-1.5h, cleaned after the completion of incubation with TBST
Repeatedly, finally developing solution is dropped evenly on pvdf membrane, is encased with masking foil and is protected from light 3-7min, pass through chemiluminescence imaging instrument
Immune result is observed, is compared with the result of true silk fabric cultural relics sample, with true silk fabric cultural relics sample closer to then emulating
It spends higher.For example, true silk fabric cultural relics sample test result are as follows: sequence is used to be immunized for the antigen of MQRKNKNHGILGK
When, fibroin content intensity is between 60 ten thousand to 150 ten thousand, when sequence being used to be immunized for the antigen of GAGAGSGAGAGS,
Intensity chooses optimal alkali ageing time 29,000,000 3,000 ten thousand or so, with this.
Preferably, the bath raio of mulberry silk and sodium carbonate degumming liquid is 1: 90-110g/mL, and heating temperature is in step 1)
95-100℃。
Preferably, it is 2000 that the specification for bag filter used of dialysing, which is molecular cut off, and dialysis procedure is lasting in step 3)
2-4 days, and a water is changed every 2-4h.
Preferably, in step 5), glue operation specifically: by separation gel with liquid-transfering gun be injected into two pieces of glass plates it
Between, flush its liquid level between then isopropanol being taken to be injected into glass plate.Then glass plate is placed in 15-20min at 35-40 DEG C；
After gelling to be separated is solid, isopropanol is outwelled, then add concentration glue and plugs comb immediately, again by device by glass plate
It is placed in 10-20min at 35-40 DEG C, the glass plate after glue is put into electrophoresis tank and carries out leak detection operation；Short glass is wanted when placement
Inwardly, long glass plate is outwardly for glass plate；Then it dials comb and forms swimming lane.
Preferably, in step 7), electrophoretic procedures process are as follows: adjust voltage to 75-85V, by voltage after electrophoresis 8-12min
110-130V is risen to, when bromophenol blue being waited to reach near bottom, stops electrophoresis.
Preferably, the antigen of primary antibody is respectively the feature peptide fragment GAGAGSGAGAGS of mulberry silk crystal region in step 9)
With the feature peptide fragment MQRKNKNHGILGK of amorphous region；It is combined above two feature peptide fragment as haptens and carrier protein
After carry out animal immune, obtain two kinds of anti-fibroin albumen sequence antibodies.
It is compared with the prior art, the beneficial effects of the present invention are:
(1) different alkali ageing times have been probed into the present invention on the immunologic influence of mulberry silk, immunoblotting can be with
The field that conventional micro- detection and infrared detection do not touch is supplemented well, preferably provides ginseng for historical relic simulation field
It examines.
(2) present invention summarizes the rule of silk aging, to establish historical relic sample and people according to artificial alkali Aging simulation mode
Connection between work aging sample in shape immune detection.
(3) the alkali aging used in the present invention is different from conventional aging method, has high treating effect, work fast
The advantages that.
(4) two kinds of not homotactic haptens, respectively the feature peptide fragment of mulberry silk crystal region have been used in the present invention
The feature peptide fragment MQRKNKNHGILGK of GAGAGSGAGAGS and amorphous region.Two kinds of antibody that not prepared by synantigen are respectively to alkali
Aging sample carries out immunology detection, the analysis alkali ageing time difference bring Immunological Changes that can more integrate.
Detailed description of the invention
Fig. 1 is that (antigen sequence is for the western blot result of different time alkali aging silk sample of the present invention
MQRKNKNHGILGK)。
Fig. 2 is that (antigen sequence is for the western blot result of different time alkali aging sample of the present invention
GAGAGSGAGAGS)。
Fig. 3 is different alkali ageing time fibroin content analysis (antigen sequence MQRKNKNHGILGK) of the invention.
Fig. 4 is different alkali ageing time fibroin content analysis (antigen sequence GAGAGSGAGAGS).
Specific embodiment
The present invention will be further described with reference to the examples below.
Embodiment 1
1) mulberry silk is immersed in first in the degumming liquid (sodium carbonate liquor) prepared, the degumming liquid bath raio of preparation is 1:
100, it is to be heated in 98 degree, and be stirred continuously in temperature, continues half 0.5h, and repeat this operation 3 times；
2) after silk gum is sufficiently sloughed, then fibroin insoluble in beaker is then placed into 60 with distilled water flushing 5 times
It is dried overnight in the baking oven of degree.It takes seven groups of samples, every group of 1.5g respectively afterwards every other day, then pours into sodium hydroxide solution respectively, bathe
Than being 1: 100, be placed in 37 degree of baking ovens, place respectively 0h, 2h, 6h, 12h, for 24 hours, 36h, 48h；
3) it after alkali ageing time reaches, is filtered, is then completely dried in being placed in 37 degree of baking ovens.Wait be completely dried
Afterwards, protein extract is added according to 1: 50 bath raio in every group of sample, and heats in the water-bath that temperature is 98 degree, and constantly dissolve
2h is stirred, then will be dialysed dissolved with the protein extract of fibroin albumen, what when dialysis used is that molecular cut off is
2000 bag filter, dialysis procedure continue 3 days, and change a water every 3h or so, and bag filter saves under 4 degree of environment always；
4) obtained silk fibroin protein solution is filtered, removes insoluble impurity.Obtained pure silk fibroin protein solution is carried out
Freeze-drying process was ground into powder after 2 days again；
5) gum-making rack wash with distilled water first, takes out after dry 15min in 60 degree of baking ovens, after putting up gum-making rack, inspection
Look into whether leak.As water-tight, subsequent glue operation is carried out, specific glue behaviour is by prepared separation gel liquid relief
Rifle being injected between two pieces of glass plates slowly, makes its liquid level neat between then taking a certain amount of isopropanol to be also injected into glass plate
It is flat.Then device is put into 37 degree of baking ovens solidifies separation gel, standing time 15min.It, will be above different after its solidification
Propyl alcohol is outwelled, and is then added prepared concentration glue and is plugged comb immediately.Just device is placed in again after insertion
After the time arrives, the glass plate for making glue is put into electrophoresis tank by 15min in 37 degree of baking ovens, this is to carry out leak detection operation again.Note
Meaning wants short slab inwardly when placing, and long slab is outwardly.Then it just dials and removes comb, slowly steadily extracted here to ensure that swimming lane shapes
Standard；
6) protein powder for the different alkali ageing times that front has made is dissolved in CB (pH=9.6), formation
Solution concentration is 10mg/mL, then respectively takes the reduced form 5XSDS sample-loading buffer of 3ul in the small test tube of 1.5mL, then plus
Enter the mixed liquid of protein of different alkali ageing times, and heat 10min in 98 degree of water-baths, then carries out centrifugal treating；
7) it takes supernatant to carry out next electrophoretic procedures, is added in first swimming lane and does not contaminate marker for molecular weight mark
Will object, then sequentially add alkali ageing time be 0h, 2h, 6h, 12h, for 24 hours, the fibroin albumen mixed liquor of 36h, 48h.Then electric
The operating process of swimming is to adjust voltage to arrive 80V, and voltage is risen to 120V after electrophoresis 10min, is waited near bromophenol blue arrival bottom
When, just stop electrophoresis, glass plate taking-up is placed in the culture dish for filling distilled water after the completion, then by glue between glass plate
It is stripped out and 3 times wash with distilled water, electrophoresis result can be observed by chemiluminescence imaging instrument after cleaning completely；
8) after having observed, the pvdf membrane of a long 9cm, width 5.5cm is first cut, 1min in methyl alcohol is then impregnated and swashed
Hair, subsequent and sponge and the blistering of filter paper one, followed by building for sandwich structure, then can in transfering buffering liquid
Transfer need to be wrapped in reaction unit surrounding because transfer process can release heat with ice bag, and Transfer current 200mA is entire to transfer
Process continues 0.5h or so；
9) after the completion of transferring, pvdf membrane is placed in chemiluminescence imaging instrument, whether detection transfer result is good.Then will
Prepared 20mL confining liquid is poured on film, outwells after shaking table closing 1h, then the primary antibody 10mL diluted with TBST is added,
Primary antibody shaking table is incubated for 1h, is then cleaned 3 times with TBST, each dosage 20mL, shaking table 10min, after cleaning 3 times, adds dilution
Secondary antibody 10mL afterwards is incubated for 1h, is equally cleaned 3 times with TBST after the completion of being incubated for, finally prepared developing solution is dropped evenly
It on film, is encased with masking foil and is protected from light 5min, immune result is then observed by chemiluminescence imaging instrument.
True silk fabric cultural relics sample test result are as follows: when sequence being used to be immunized for the antigen of MQRKNKNHGILGK, silkworm
Silk-fibroin content intensity is between 60 ten thousand to 150 ten thousand, and when sequence being used to be immunized for the antigen of GAGAGSGAGAGS, intensity exists
29000000 3,000 ten thousand or so.
After being detected according to the method for embodiment 1, testing result is as shown in Figures 1 to 4.
As shown in Figs 1-4, it can be readily apparent that from Fig. 1 from 2h to 12h with the raising of alkali ageing time, band
Color is gradually faded away, and illustrates that silk palliating degradation degree is heavier, though subsequent band color change is unobvious, by Fig. 4
Quantification of intensities analysis is it will be seen that intensity is reduced with the increase of alkali ageing time, never the 16000000 of alkali aging
It nearby arrives near the 2000000 of alkali aging 48h, declines degree clearly, although from alkali aging 0h to the process of alkali aging 2h
In, intensity value has certain ascendant trend, but this may be the deviation present in the quantitative analysis of intensity.And totally come
It sees, increase with alkali ageing time, intensity downward trend is very significant.After having changed a kind of primary antibody, band color change in Fig. 2
It is not obvious enough, but pass through quantification of intensities analysis in Fig. 4, it can be seen that from alkali aging 0h to alkali aging 48h, intensity value is decline
, this is also consistent with the conclusion in Fig. 1.Although in the intensity value in Fig. 4 there is also it is certain repeatedly, overall trend still conforms to
Alkali ageing time and this proportional conclusion of palliating degradation degree, also illustrate that two kinds of antibody can detected this changed
Journey.Furthermore the protein degradation of crystal region is at the uniform velocity degraded with the increase of alkali ageing time, and the protein degradation of amorphous region is first
The degradation trend delayed after urgency.The corresponding sequence antigen for finally reference being combined to provide obtains antibody and carries out the silkworm obtained after being immunized
Silk-fibroin intensity, historical relic alkali aging Best Times are between 2-6h.
Embodiment 2
1) mulberry silk is immersed in first in the degumming liquid (sodium carbonate liquor) prepared, the degumming liquid bath raio of preparation is 1:
100, it is to be heated in 98 degree, and be stirred continuously in temperature, continues half 0.5h, and repeat this operation 4 times；
2) after silk gum is sufficiently sloughed, then fibroin insoluble in beaker is then placed into 60 with distilled water flushing 5 times
It is dried overnight in the baking oven of degree.It takes seven groups of samples, every group of 1.5g respectively afterwards every other day, then pours into sodium hydroxide solution respectively, bathe
Than being 1: 100, be placed in 36 degree of baking ovens, place respectively 0h, 2h, 6h, 12h, for 24 hours, 36h, 48h；
3) it after alkali ageing time reaches, is filtered, is then completely dried in being placed in 37 degree of baking ovens.Next to complete
After white drying, protein extract is added according to 1: 50 bath raio in every group of sample, and heats in the water-bath that temperature is 96 degree, not
Disconnected dissolution stirring 2.5h, then dialyses the protein extract dissolved with fibroin albumen, and what when dialysis used is retention point
The bag filter that son amount is 2000, dialysis procedure continues 4 days, and changes a water every 5h or so, and bag filter is always under 4 degree of environment
It saves；
4) obtained silk fibroin protein solution is filtered, removes insoluble impurity.Obtained pure silk fibroin protein solution is carried out
Freeze-drying process was ground into powder after 2 days again；
5) gum-making rack wash with distilled water first, takes out after dry 20min in 60 degree of baking ovens, after putting up gum-making rack, inspection
Look into whether leak.As water-tight, subsequent glue operation is carried out, specific glue behaviour is by prepared separation gel liquid relief
Rifle being injected between two pieces of glass plates slowly, makes its liquid level neat between then taking a certain amount of isopropanol to be also injected into glass plate
It is flat.Then device is put into 37 degree of baking ovens solidifies separation gel, standing time 20min.It, will be above different after its solidification
Propyl alcohol is outwelled, and is then added prepared concentration glue and is plugged comb immediately.Just device is placed in again after insertion
After the time arrives, the glass plate for making glue is put into electrophoresis tank by 15min in 37 degree of baking ovens, this is to carry out leak detection operation again.Note
Meaning wants short slab inwardly when placing, and long slab is outwardly.Then it just dials and removes comb, slowly steadily extracted here to ensure that swimming lane shapes
Standard；
6) protein powder for the different alkali ageing times that front has made is dissolved in CB (pH=9.6), formation
Solution concentration is 10mg/mL, then respectively takes the reduced form 5XSDS sample-loading buffer of 3ul in the small test tube of 1.5mL, then plus
Enter the mixed liquid of protein of different alkali ageing times, and heat 12min in 95 degree of water-baths, then carries out centrifugal treating；
7) it takes supernatant to carry out next electrophoretic procedures, is added in first swimming lane and does not contaminate marker for molecular weight mark
Will object, then sequentially add alkali ageing time be 0h, 2h, 6h, 12h, for 24 hours, the fibroin albumen mixed liquor of 36h, 48h.Then electric
The operating process of swimming is to adjust voltage to arrive 80V, and voltage is risen to 120V after electrophoresis 10min, is waited near bromophenol blue arrival bottom
When, just stop electrophoresis, glass plate taking-up is placed in the culture dish for filling distilled water after the completion, then by glue between glass plate
It is stripped out and 4 times wash with distilled water, electrophoresis result can be observed by chemiluminescence imaging instrument after cleaning completely；
8) after having observed, the pvdf membrane of a long 9cm, width 5.5cm is first cut, 1min in methyl alcohol is then impregnated and swashed
Hair, subsequent and sponge and the blistering of filter paper one, followed by building for sandwich structure, then can in transfering buffering liquid
Transfer need to be wrapped in reaction unit surrounding because transfer process can release heat with ice bag, and Transfer current 200mA is entire to transfer
Process continues 0.5h or so；
9) after the completion of transferring, pvdf membrane is placed in chemiluminescence imaging instrument, whether detection transfer result is good.Then will
Prepared 20mL confining liquid is poured on film, outwells after shaking table closing 1h, then the primary antibody 10mL diluted with TBST is added,
Primary antibody shaking table is incubated for 1h, is then cleaned 3 times with TBST, each dosage 20mL, shaking table 10min, after cleaning 3 times, adds dilution
Secondary antibody 10mL afterwards is incubated for 1h, is equally cleaned 3 times with TBST after the completion of being incubated for, finally prepared developing solution is dropped evenly
It on film, is encased with masking foil and is protected from light 5min, then can observe immune result by chemiluminescence imaging instrument.
Embodiment 3
1) mulberry silk is immersed in first in the degumming liquid (sodium carbonate liquor) prepared, the degumming liquid bath raio of preparation is 1:
100, it is to be heated in 98 degree, and be stirred continuously in temperature, continues half 0.5h, and repeat this operation 5 times；
2) after silk gum is sufficiently sloughed, then fibroin insoluble in beaker is then placed into 60 with distilled water flushing 5 times
It is dried overnight in the baking oven of degree.It takes seven groups of samples, every group of 1.5g respectively afterwards every other day, then pours into sodium hydroxide solution respectively, bathe
Than being 1: 100, be placed in 37 degree of baking ovens, place respectively 0h, 2h, 6h, 12h, for 24 hours, 36h, 48h；
3) it after alkali ageing time reaches, is filtered, is then completely dried in being placed in 37 degree of baking ovens.Next to complete
After white drying, protein extract is added according to 1: 50 bath raio in every group of sample, and heats in the water-bath that temperature is 99 degree, not
Disconnected dissolution stirring 3h, then dialyses the protein extract dissolved with fibroin albumen, and what when dialysis used is retention molecule
The bag filter that amount is 2000, dialysis procedure continues 3 days, and changes a water every 4h or so, and bag filter is protected under 4 degree of environment always
It deposits；
4) obtained silk fibroin protein solution is filtered, removes insoluble impurity.Obtained pure silk fibroin protein solution is carried out
Freeze-drying process was ground into powder after 2 days again；
5) gum-making rack wash with distilled water first, takes out after dry 16min in 60 degree of baking ovens, after putting up gum-making rack, inspection
Look into whether leak.As water-tight, subsequent glue operation is carried out, specific glue behaviour is by prepared separation gel liquid relief
Rifle being injected between two pieces of glass plates slowly, makes its liquid level neat between then taking a certain amount of isopropanol to be also injected into glass plate
It is flat.Then device is put into 37 degree of baking ovens solidifies separation gel, standing time 18min.It, will be above different after its solidification
Propyl alcohol is outwelled, and is then added prepared concentration glue and is plugged comb immediately.Just device is placed in again after insertion
After the time arrives, the glass plate for making glue is put into electrophoresis tank by 15min in 37 degree of baking ovens, this is to carry out leak detection operation again.Note
Meaning wants short slab inwardly when placing, and long slab is outwardly.Then it just dials and removes comb, slowly steadily extracted here to ensure that swimming lane shapes
Standard；
6) protein powder for the different alkali ageing times that front has made is dissolved in CB (pH=9.6), formation
Solution concentration is 10mg/mL, then respectively takes the reduced form 5XSDS sample-loading buffer of 3ul in the small test tube of 1.5mL, then plus
Enter the mixed liquid of protein of different alkali ageing times, and heat 15min in 99 degree of water-baths, then carries out centrifugal treating；
7) it takes supernatant to carry out next electrophoretic procedures, is added in first swimming lane and does not contaminate marker for molecular weight mark
Will object, then sequentially add alkali ageing time be 0h, 2h, 6h, 12h, for 24 hours, the fibroin albumen mixed liquor of 36h, 48h.Then electric
The operating process of swimming is to adjust voltage to arrive 80V, and voltage is risen to 120V after electrophoresis 10min, is waited near bromophenol blue arrival bottom
When, just stop electrophoresis, glass plate taking-up is placed in the culture dish for filling distilled water after the completion, then by glue between glass plate
It is stripped out and 5 times wash with distilled water, electrophoresis result can be observed by chemiluminescence imaging instrument after cleaning completely；
8) after having observed, the pvdf membrane of a long 9cm, width 5.5cm is first cut, 1min in methyl alcohol is then impregnated and swashed
Hair, subsequent and sponge and the blistering of filter paper one, followed by building for sandwich structure, then can in transfering buffering liquid
Transfer need to be wrapped in reaction unit surrounding because transfer process can release heat with ice bag, and Transfer current 200mA is entire to transfer
Process continues 0.5h or so；
9) after the completion of transferring, pvdf membrane is placed in chemiluminescence imaging instrument, whether detection transfer result is good.Then will
Prepared 20mL confining liquid is poured on film, outwells after shaking table closing 1h, then the primary antibody 10mL diluted with TBST is added,
Primary antibody shaking table is incubated for 1h, is then cleaned 3 times with TBST, each dosage 20mL, shaking table 10min, after cleaning 3 times, adds dilution
Secondary antibody 10mL afterwards is incubated for 1h, is equally cleaned 3 times with TBST after the completion of being incubated for, finally prepared developing solution is dropped evenly
It on film, is encased with masking foil and is protected from light 5min, immune result is then observed by chemiluminescence imaging instrument.
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 (6)
1. a kind of method based on Western blot silk fabric cultural relics simulation sample fidelity, it is characterised in that including walking as follows
It is rapid:
1) mulberry silk is immersed in sodium carbonate degumming liquid, heats and be stirred continuously 20-40min, it is multiple to repeat this operation；
2) after silk gum is sufficiently sloughed, insoluble fibroin distilled water flushing is multiple, it is subsequently placed in drying at 50-70 DEG C,
Then gained fibroin is divided into multiple groups, is added respectively into sodium hydroxide solution at 35-40 DEG C by bath raio 1: 90-110g/mL
Alkali aging different time；
3) it is filtered after alkali aging, gained fibroin is placed in drying, every group of fibroin at 35-40 DEG C and presses bath raio 1: 40- respectively
60g/mL is added into protein extract, the heating stirring 1.5-2.5h in 95-100 DEG C of water-bath, then will be dissolved with fibroin egg
White protein extract is dialysed；
4) protein extract obtained by step 3) is filtered, freeze-drying pulverizes after 1-3 days, obtains silk fibroin powder；
5) gum-making rack wash with distilled water takes out after dry 10-20min at 55-65 DEG C, after putting up gum-making rack, checks whether
Leak carries out subsequent glue operation, obtained tundish accompanies two pieces of glass plates of several swimming lanes as water-tight；
6) each group silk fibroin powder is dissolved separately in and the fibroin albumen mixed liquor that concentration is 8-12mg/mL is made in CB liquid,
Then it respectively takes the reduced form 5XSDS sample-loading buffer of 2.5-3.5ul in test tube, is then respectively adding the mixing of each group fibroin albumen
Liquid heats 8-12min, centrifugal treating in 95-100 DEG C of water-bath；
7) take supernatant carry out electrophoretic procedures, be added in first swimming lane do not contaminate marker be molecular weight marker, then according to
Secondary addition each group supernatant carries out electrophoresis, by the glue removing between glass plate and 3 to 4 times wash with distilled water after the completion of electrophoresis,
Electrophoresis result is observed by chemiluminescence imaging instrument after cleaning；
8) after observing, a pvdf membrane is cut, 0.5-1.5min in methanol is soaked in and excited, then by pvdf membrane and sponge
Filter paper is dipped in transfering buffering liquid, is carried out building for sandwich structure, is then transferred, is wrapped up in transfer process with ice bag
In reaction unit surrounding, Transfer current 190-210mA, transfer process continues 20-40min；
9) 18-22mL confining liquid is poured on pvdf membrane, is outwelled after shaking table closing 0.5-1.5h, 8-12mL is added and is diluted with TBST
Good primary antibody, primary antibody shaking table are incubated for 0.5-1.5h, are then cleaned repeatedly with TBST, each dosage 18-22mL, shaking table 8-12min,
After cleaning repeatedly, adds the secondary antibody after 8-12mL dilutes and be incubated for 0.5-1.5h, cleaned repeatedly, most after the completion of incubation with TBST
Developing solution is dropped evenly on pvdf membrane afterwards, is encased with masking foil and is protected from light 3-7min, is exempted from by the observation of chemiluminescence imaging instrument
Epidemic disease is higher closer to then fidelity with true silk fabric cultural relics sample as a result, compare with the result of true silk fabric cultural relics sample.
2. method as described in claim 1, which is characterized in that in step 1), the bath raio of mulberry silk and sodium carbonate degumming liquid is 1:
90-110g/mL, heating temperature are 95-100 DEG C.
3. method as described in claim 1, which is characterized in that in step 3), the specification for bag filter used of dialysing is retention molecule
Amount is 2000, and dialysis procedure continues 2-4 days, and changes a water every 2-4h.
4. method as described in claim 1, which is characterized in that in step 5), glue operation specifically: by separation gel liquid-transfering gun
It is injected between two pieces of glass plates, flushes its liquid level between then isopropanol being taken to be injected into glass plate；Then glass plate is placed in
15-20min at 35-40 DEG C；After gelling to be separated is solid, isopropanol is outwelled, then add concentration glue and plugs comb immediately,
Glass plate is placed in 10-20min at 35-40 DEG C by device again, the glass plate after glue is put into electrophoresis tank and is hunted leak
Operation；Short glass plate is wanted when placement, and long glass plate is outwardly inwardly；Then it dials comb and forms swimming lane.
5. method as described in claim 1, which is characterized in that in step 7), electrophoretic procedures process are as follows: adjust voltage to 75-
Voltage is risen into 110-130V after 85V, electrophoresis 8-12min, when bromophenol blue being waited to reach near bottom, stops electrophoresis.
6. method as described in claim 1, which is characterized in that in step 9), the antigen of primary antibody is respectively mulberry silk crystal region
The feature peptide fragment MQRKNKNHGILGK of feature peptide fragment GAGAGSGAGAGS and amorphous region；Using above two feature peptide fragment as
Haptens and carrier protein carry out animal immune after combining, and obtain two kinds of anti-fibroin albumen sequence antibodies.
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Application publication date: 20191105 |