CN108872585A - A kind of detection method of collagen end peptide - Google Patents
A kind of detection method of collagen end peptide Download PDFInfo
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- CN108872585A CN108872585A CN201810440528.9A CN201810440528A CN108872585A CN 108872585 A CN108872585 A CN 108872585A CN 201810440528 A CN201810440528 A CN 201810440528A CN 108872585 A CN108872585 A CN 108872585A
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- 108010035532 Collagen Proteins 0.000 title claims abstract description 89
- 102000008186 Collagen Human genes 0.000 title claims abstract description 89
- 229920001436 collagen Polymers 0.000 title claims abstract description 85
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 56
- 238000001514 detection method Methods 0.000 title claims abstract description 34
- 238000012546 transfer Methods 0.000 claims abstract description 31
- 238000011534 incubation Methods 0.000 claims abstract description 24
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 18
- 238000001962 electrophoresis Methods 0.000 claims abstract description 16
- 239000007788 liquid Substances 0.000 claims abstract description 13
- 239000012153 distilled water Substances 0.000 claims abstract description 7
- 238000011161 development Methods 0.000 claims abstract description 3
- 239000000523 sample Substances 0.000 claims description 49
- 239000000872 buffer Substances 0.000 claims description 11
- 239000000243 solution Substances 0.000 claims description 11
- 238000002156 mixing Methods 0.000 claims description 10
- 230000009514 concussion Effects 0.000 claims description 8
- 239000000047 product Substances 0.000 claims description 8
- 238000004458 analytical method Methods 0.000 claims description 7
- 239000007853 buffer solution Substances 0.000 claims description 7
- 239000012723 sample buffer Substances 0.000 claims description 7
- 239000006228 supernatant Substances 0.000 claims description 7
- 239000000020 Nitrocellulose Substances 0.000 claims description 6
- 102000004160 Phosphoric Monoester Hydrolases Human genes 0.000 claims description 6
- 108090000608 Phosphoric Monoester Hydrolases Proteins 0.000 claims description 6
- 239000003513 alkali Substances 0.000 claims description 6
- 238000009835 boiling Methods 0.000 claims description 6
- 229920001220 nitrocellulos Polymers 0.000 claims description 6
- 239000013595 supernatant sample Substances 0.000 claims description 6
- 239000002585 base Substances 0.000 claims description 5
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 claims description 5
- 239000007787 solid Substances 0.000 claims description 5
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims description 4
- 239000004202 carbamide Substances 0.000 claims description 4
- 238000004090 dissolution Methods 0.000 claims description 4
- DNJIEGIFACGWOD-UHFFFAOYSA-N ethyl mercaptane Natural products CCS DNJIEGIFACGWOD-UHFFFAOYSA-N 0.000 claims description 4
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 claims description 4
- 238000005119 centrifugation Methods 0.000 claims description 2
- BAVYZALUXZFZLV-UHFFFAOYSA-N Methylamine Chemical compound NC BAVYZALUXZFZLV-UHFFFAOYSA-N 0.000 claims 1
- 238000000034 method Methods 0.000 abstract description 20
- 230000005847 immunogenicity Effects 0.000 abstract description 6
- 239000003292 glue Substances 0.000 description 15
- 238000000926 separation method Methods 0.000 description 12
- 238000000605 extraction Methods 0.000 description 10
- 102000004169 proteins and genes Human genes 0.000 description 7
- 108090000623 proteins and genes Proteins 0.000 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 description 6
- 238000011068 loading method Methods 0.000 description 6
- 102000004190 Enzymes Human genes 0.000 description 5
- 108090000790 Enzymes Proteins 0.000 description 5
- 229940088598 enzyme Drugs 0.000 description 5
- 238000003119 immunoblot Methods 0.000 description 5
- 230000000890 antigenic effect Effects 0.000 description 4
- 230000029087 digestion Effects 0.000 description 4
- 230000005611 electricity Effects 0.000 description 4
- 238000004064 recycling Methods 0.000 description 4
- 235000020183 skimmed milk Nutrition 0.000 description 4
- 238000001262 western blot Methods 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 210000004027 cell Anatomy 0.000 description 3
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 3
- 101710096389 Collagen alpha chain Proteins 0.000 description 2
- 239000000427 antigen Substances 0.000 description 2
- 102000036639 antigens Human genes 0.000 description 2
- 108091007433 antigens Proteins 0.000 description 2
- 239000012472 biological sample Substances 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 239000000515 collagen sponge Substances 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 230000028993 immune response Effects 0.000 description 2
- 230000003287 optical effect Effects 0.000 description 2
- 239000013558 reference substance Substances 0.000 description 2
- 238000002271 resection Methods 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- QCVGEOXPDFCNHA-UHFFFAOYSA-N 5,5-dimethyl-2,4-dioxo-1,3-oxazolidine-3-carboxamide Chemical compound CC1(C)OC(=O)N(C(N)=O)C1=O QCVGEOXPDFCNHA-UHFFFAOYSA-N 0.000 description 1
- QRXMUCSWCMTJGU-UHFFFAOYSA-N 5-bromo-4-chloro-3-indolyl phosphate Chemical compound C1=C(Br)C(Cl)=C2C(OP(O)(=O)O)=CNC2=C1 QRXMUCSWCMTJGU-UHFFFAOYSA-N 0.000 description 1
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 1
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 1
- 101800001415 Bri23 peptide Proteins 0.000 description 1
- 101800000655 C-terminal peptide Proteins 0.000 description 1
- 102400000107 C-terminal peptide Human genes 0.000 description 1
- 102000002322 Egg Proteins Human genes 0.000 description 1
- 108010000912 Egg Proteins Proteins 0.000 description 1
- 241001062009 Indigofera Species 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 102000057297 Pepsin A Human genes 0.000 description 1
- 108090000284 Pepsin A Proteins 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- JDZJVWAHZYIHFA-UHFFFAOYSA-N [Br].C1(=CC=CC=C1)O Chemical compound [Br].C1(=CC=CC=C1)O JDZJVWAHZYIHFA-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 210000004899 c-terminal region Anatomy 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000000326 densiometry Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 235000014103 egg white Nutrition 0.000 description 1
- 210000000969 egg white Anatomy 0.000 description 1
- 235000013601 eggs Nutrition 0.000 description 1
- 210000002744 extracellular matrix Anatomy 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 230000023597 hemostasis Effects 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000002163 immunogen Effects 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 229940111202 pepsin Drugs 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 238000007781 pre-processing Methods 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000002203 pretreatment Methods 0.000 description 1
- 230000001172 regenerating effect Effects 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000009182 swimming Effects 0.000 description 1
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- 238000005303 weighing Methods 0.000 description 1
Classifications
-
- 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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
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- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Immunology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a kind of detection methods of collagen end peptide, including step：(1) sample treatment and electrophoresis；(2) albumen transfers；(3) closing and incubation：Transfer film obtained by step (2) is taken, is put into confining liquid and shakes；Then collagen end peptide antibody is added, primary antibody incubation is carried out to transfer film, secondary antibody is added later and is incubated for；(4) chromogenic reaction：Transfer film after taking step (3) secondary antibody to be incubated for is added chromophoric solution colour developing and adds distilled water until band occurs and reacted with color development stopping, the type of collagen end peptide in sample to be tested is judged according to gained band.The present invention provides it is a kind of can effectively qualitatively, sxemiquantitative detection Exogenous Collagen albumen middle-end peptide content method, be Exogenous Collagen albumen base biological product immunogenicity detect providing method foundation.
Description
Technical field
The present invention relates to collagen detection technique field, especially a kind of detection method of collagen end peptide.
Background technique
Hemostasis, reparation, defect filling of the Exogenous Collagen albumen base biology after tissue trauma, and for group weaver
Engineering support and play great function in the regenerative medicine that grows up.Although collagen is generally considered safe, tool
There is a multi-functional biomaterial, but as it applies more and more extensive in organizational project, collagen clinically may
Caused immune response is concerned.The antigenic determinant of Exogenous Collagen albumen is broadly divided into three classifications：1. complete helix
The antigenic determinant of structure, the less immunogenic of this part；2. the antigenic determinant in coiled strand, only when collagen
After triple-helix structure is destroyed, immunogenicity is just shown；3. the antigenic determinant of C- and N- (being referred to as collagen end peptide), this
Partial immunogenicity is considered as the maximum reason that collagen causes immune response.In general, in collagen extraction process
In, collagen end peptide can be removed using enzyme, but digestion is but difficult to judge except the effect of end peptide.In addition, for some
De- cell products, it is even more to be difficult to judge that wherein whether the end peptide of collagen, which also retains,.
Currently, having had already appeared the enzyme linked immunological kit at the end collagen C- and N- on the market, it to be used for quantitative detection blood
Clearly, the content of blood, tissue homogenate and related liquid sample middle-end peptide.Due to the Exogenous Collagen albumen or de- cell of extraction
System existing for product collagen is more complex, and (such as the collagen system of extraction is containing end peptide, the foreign protein after enzyme, digestion
Deng；For another example taking off cell products collagen, there are other extracellular matrixs；), meeting Interference Detection be easy to cause false positive, false yin
Property etc. is as a result, stability is poor.Therefore, the judgement for the collagen end peptide content that the not applicable judgement of these methods is extracted.
Summary of the invention
Based on the above issues, providing it is an object of the invention to overcome in place of above-mentioned the deficiencies in the prior art a kind of can have
The method of qualitative, sxemiquantitative the detection Exogenous Collagen albumen middle-end peptide content of effect, is Exogenous Collagen albumen base biological production
The immunogenicity of product detects providing method foundation.
To achieve the above object, the technical scheme adopted by the invention is as follows：A kind of detection method of collagen end peptide, including
Following steps：
(1) sample treatment and electrophoresis：After mixing by sample to be tested and SDS buffer, centrifugation obtains Supernatant samples；
It takes the Supernatant samples to carry out SDS-PAGE electrophoresis, is recycled after electrophoresis, obtain purpose gel；
(2) albumen transfers：Nitrocellulose filter, filter paper and step (1) resulting gel are put down in transferring film buffer solution
Weighing apparatus 20~30 minutes, carries out transferring film later, obtains transfer film；
(3) closing and incubation：Transfer film obtained by step (2) is taken, is put into confining liquid and shakes；Then collagen end is added
Peptide antibody carries out primary antibody incubation to transfer film, and secondary antibody is added later and is incubated for；
(4) chromogenic reaction：Chromophoric solution colour developing is added in transfer film after taking step (3) secondary antibody to be incubated for, until band occurs,
It adds distilled water to react with color development stopping, the type of collagen end peptide in sample to be tested is judged according to gained band.
It should be noted that SDS-PAGE electrophoresis preferably 6% -8% separation gel and 4% concentration glue, preferably 10 when electrophoresis~
20mA electric current, electrophoresis time is 20~30min, and after the bromophenol blue in loading sample enters separation gel, adjustment electrophoretic current is
20-40min stops electrophoresis after bromophenol blue reaches separation gel bottom；When albumen transfers, filter paper is preferably 6, and transferring film electric current is excellent
It is selected as 400~450mA, the use of ice bath control transferring film temperature is 4-15 DEG C, the transferring film time is preferably 80-120min；In closing,
Transfer film closes 10-12h at 2-3h or 4 DEG C of concussion closing at 20-30 DEG C in confining liquid；When primary antibody is incubated for, condition is preferred
For 1-2h or 4 DEG C of incubation 10-12h of 37 DEG C of incubations, when secondary antibody is incubated for, incubation conditions are preferably 37 DEG C of 1.5-3h；Developing time is excellent
Select 3~8min；Preferably, the confining liquid is the skimmed milk power that mass concentration is 5%.
Preferably, the primary antibody is the end ox I-type collagen C- and/or the end N- antibody；It is highly preferred that the primary antibody is dilute
Releasing multiple is 1：100~200.
Preferably, the secondary antibody is the anti-rabbit IgG antibody of alkali phosphatase enzyme mark；It is highly preferred that the dilution of the secondary antibody
Multiple is 1：5000~20000.
Preferably, the sample to be tested is the biological sample or product of Exogenous Collagen albumen.It should be noted that this hair
Bright detection method can also detect endogenous or other collagen eggs in addition to can detecte Exogenous Collagen protein biology sample
White biological sample.
Preferably, the detection method further includes step (5)：It is anti-using colour developing in photo densitometry analytical procedure (4)
Transfer film after should terminating, to judge the content of collagen end peptide in sample to be tested.It should be noted that using step (5)
It can detecte the C-terminal peptide of collagen and/or the content of N-terminal peptide.
Preferably, in the step (1), when sample to be tested be extract collagen liquid when, can directly with SDS buffer
Mixing；When sample to be tested is solid, first solid sample is mixed with sample buffer, at room temperature concussion dissolution 4h,
It is centrifuged 5min under 5000rmp, obtains supernatant；Again by the supernatant or collagen liquid and SDS buffer according to 1:1 volume
Than handling 3~6min in 100 DEG C of boiling water baths after mixing, after being cooled to room temperature, at 4 DEG C, it is centrifuged 5min under 5000rmp, obtains
Supernatant samples.By the step process, protein chain can be promoted to crack in complex system, dissolve collagen sufficiently, by from
Heart separation obtains the sample to upper electrophoresis.
Preferably, 100mM trishydroxymethylaminomethane, 8M urea, 2%SDS and 2% are contained in the sample buffer
Mercaptoethanol, the pH value of the sample buffer are 8.0.As a result, the sample buffer can make in complex system collagen with
The cross-bond of other substances destroys, the dissolution of collagen in promotion system, in order to subsequent analysis.
In conclusion beneficial effects of the present invention are：
1, Exogenous Collagen albumen middle-end peptide content can effectively qualitative, sxemiquantitative be detected the present invention provides a kind of
Method is that the immunogenicity of Exogenous Collagen albumen base biological product detects providing method foundation；
2, compared to the Enzyme-multiplied immune technique of existing detection collagen end peptide, the present invention uses immunoblotting combination light
Density analysis method detects Exogenous Collagen albumen end peptide content, and the antigen of identification collagen end peptide that can be specific determines
Cluster, it is unrelated with end peptide integrality (i.e. end peptide needs not be complete)；
3, detection method of the invention can apply to system complicated (as containing other foreign proteins, polysaccharide, fat, enzyme system)
Sample to be tested greatly improves detectability range by pre-processing sample to be tested；This method is not by the shadow of system complexity
It rings, accurately obtains effectively holding that peptide is qualitative, semi-quantitative results by the analysis of purpose band.
Detailed description of the invention
Fig. 1 is the western blot figure of the end ox I-type collagen C- antigentic specificity band；
Fig. 2 is the immunoblotting map of the end collagen α chain N- different extraction times；
Fig. 3 is the photodensitometry figure of Fig. 2 antigentic specificity band, and the unit of molecular weight is kda；
Fig. 4 is the immunoblotting map of the end collagen α chain C- different extraction times；
Fig. 5 is the photodensitometry figure of Fig. 4 antigentic specificity band, and the unit of molecular weight is kda；
Fig. 6 is the western blot figure of the end ox I-type collagen sponge N- antigentic specificity band.
Specific embodiment
The present invention relates to technical field of biological, more specifically, being related to a kind of utilization detected by Western blot judgement
The detection method of the content of Exogenous Collagen albumen end peptide.An object of the present invention be to provide it is a kind of can effectively qualitatively, half
The method of quantitative detection Exogenous Collagen albumen middle-end peptide content, is the immunogenicity of Exogenous Collagen albumen base biological product
Detect providing method foundation.This method combines collagen end in photodensitometry detection architecture using the side of protein immunoblot
Peptide judges containing for collagen end peptide by immune labeled specificity using the collagen in gel electrophoresis separation system
Amount.
Unless otherwise instructed, the reagent concentration in the application is mass concentration.Marker used in the present invention is purchased from
The green skies；Secondary antibody is purchased from Sigma company.I-type collagen C- and N- end antibody has purchased from Ai Bimate biological medicine (Shanghai)
Limit company；BCIP/NBT alkaline phosphatase colour reagent box is purchased from the green skies.Pay attention to：The present invention is not limited to case is embodied
The detection of ox I-type collagen end peptide purchase the end peptide antibody mutually coped with i.e. if you need to detect the collagen in other sources
It can.
It is of the invention that detection method includes the following steps：
(1) by sample to be tested and SDS buffer according to 1:3-6min is handled in 100 DEG C of boiling water baths after the mixing of 1 volume ratio, it is cold
But to after room temperature, at 4 DEG C, it is centrifuged 5min under 5000rmp, obtains loading sample；By the sample in SDS-PAGE running gel
Middle carry out electrophoresis, SDS-PAGE running gel are to be prepared and obtained with 6% -8% separation gel and 4% concentration glue；In the electricity of 10-20mA
Electrophoresis 20-30min is flowed down, after the bromophenol blue in loading sample enters separation gel, adjustment electrophoretic current is 20-40min, to bromine
Phenol indigo plant stops electrophoresis after reaching separation gel bottom；
(2) albumen transfer step：The glue of nitrocellulose filter, 6 filter paper and recycling is balanced in transferring film buffer solution
20-30min；Transferring film step is carried out with the film after balance, transferring film electric current is 400-450mA, is using ice bath control transferring film temperature
4-15 DEG C, time 80-120min, i.e. acquisition transfer film；
(3) closing and incubation step：By transfer film, concussion is closed at 2-3h or 4 DEG C at 20-30 DEG C in confining liquid
Close 10-12h；Primary antibody incubation is carried out to transfer film using collagen end peptide antibody, condition is：37 DEG C are incubated for 1-2h or 4 DEG C
It is incubated for 10-12h；Secondary antibody is to be incubated for the anti-rabbit IgG of alkali phosphatase enzyme mark, and condition is：37℃1.5-3h；
(4) chromogenic reaction is carried out：Antibody incubation film is placed in the chromophoric solution of working concentration the 3-8min that develops the color to band
Occur, distilled water terminates reaction.
Optionally, the method includes carrying out following pre-treatment step to sample to be tested：
For the collagen liquid of extraction, can directly be mixed with SDS buffer；It, can be slow with sample for solid kind sample
Rush solution (100mM trishydroxymethylaminomethane, 8M urea, 2%SDS, 2% mercaptoethanol, pH8.0) mixing, at room temperature, concussion
4h is dissolved, 5min is centrifuged at 5000rmp, takes supernatant spare.
Optionally, the collagen end peptide antibody extension rate is 1:100-1:200.
Optionally, the anti-rabbit IgG extension rate of the alkali phosphatase enzyme mark is 1:5000-1:20000.
Optionally, collagen end peptide antibody includes C- and N- antibody, using image analysis software to the light of band
Density is analyzed, and is compared with positive reference substance, and then judge the content of collagen end peptide.
Optionally, to be measured the method also includes being determined according to specific antigen band (i.e. the band of chromogenic reaction acquisition)
The type of sample collagen end peptide.
To better illustrate the object, technical solutions and advantages of the present invention, below in conjunction with the drawings and specific embodiments pair
The present invention is described further.
The detection of the 1 N of end I-type collagen C- antigentic specificity band of embodiment
A kind of embodiment of the detection method of collagen peptide end peptide of the invention, includes the following steps：
Sample treatment：By 5mL sample to be tested, (collagen of the peptide containing end, collagen as shown in figure 1, collagen contain
Justice is collagen), the 5mL negative control sample collagen of end peptide (removal) mixed respectively with 5mL SDS buffer after
100 DEG C of boiling water baths handle 3min, after being cooled to room temperature, at 4 DEG C, 5min are centrifuged under 5000rmp, it is spare to obtain loading sample；
Configure 6.5% separation gel and 4% concentration glue；Every hole applied sample amount is 10uL, runs 30min under the electric current of 10mA, in sample
After bromophenol blue enters separation gel, adjustment electrophoretic current is 20min.
Transfer：The glue of nitrocellulose filter, 6 filter paper and recycling is balanced into 30min in transferring film buffer solution.Transferring film electricity
Flow control is 4-15 DEG C using ice bath control transferring film temperature, the transferring film time is 90min in 450mA.
Closing and incubation：By transfer film, 2h is closed in concussion at 25 DEG C in 5% skimmed milk power；Followed by ox I type glue
Former PROTEIN C-end antibody carries out primary antibody incubation to transfer film, and condition is：4 DEG C of incubation 12h；Then resisting with alkali phosphatase enzyme mark
Rabbit igg is incubated for as secondary antibody, and condition is：37 DEG C, 2h.
Chromogenic reaction：Antibody incubation film is placed in chromophoric solution to the 6min that develops the color to occur to band, distilled water terminates reaction.
As a result as shown in Figure 1, using showing method of the invention to can determine that collagen that there are also the positions of the end C- chain band
And length.
The excision measure of merit at the ox end I-type collagen C- and the end N- under the different extraction times of embodiment 2
Sample description：About 200g Cowhells is taken, is added containing 15g pepsin, concentration 0.5M, volume is that the acetic acid of 1L is molten
In liquid, after mixing evenly, after standing 14h at 4 DEG C, it is placed in electric blender stirring at 4 DEG C and extracts 96h, break time
It takes the collagen mixture after extracting to be centrifuged 20min under 10000rmp speed, is used for protein immunoblot (i.e. western
Blot it) analyzes.
A kind of embodiment of the detection method of collagen peptide end peptide of the invention, includes the following steps：
Sample treatment：In 100 DEG C of boiling water baths after the 5mL sample to be tested of sampling time point is mixed with 5mL SDS buffer
3min is handled, after being cooled to room temperature, at 4 DEG C, 5min is centrifuged under 5000rmp, it is spare to obtain loading sample；6.5% point of configuration
From glue and 4% concentration glue；Every hole applied sample amount is 10uL, and 30min is run under the electric current of 10mA, and the bromophenol blue in sample, which enters, divides
After glue, adjustment electrophoretic current is 20min.
Transfer：The glue of nitrocellulose filter, 6 filter paper and recycling is balanced into 30min in transferring film buffer solution.Transferring film electricity
Flow control is 4-15 DEG C using ice bath control transferring film temperature, the transferring film time is 90min in 450mA.
Closing and incubation：By transfer film, 2h is closed in concussion at 25 DEG C in 5% skimmed milk power；Followed by ox I type glue
Former PROTEIN C-end and the end N- antibody carry out primary antibody incubation to transfer film respectively, and condition is：4 DEG C of incubation 12h；Secondary antibody is with alkaline phosphorus
The anti-rabbit IgG of sour enzyme label is incubated for, and condition is：37 DEG C, 2h.
Chromogenic reaction：Antibody incubation film is placed in chromophoric solution to the 7min that develops the color to occur to band, distilled water terminates reaction.
As a result it indicates：It is analyzed with optical density of the Quantity one software to band, is compared with positive reference substance, calculated
Relative optical density out, to be converted into the opposite resection rate of end peptide.
As a result as shown in Figure 2-5, can be found in conjunction with the method for photodensitometry, with the increase of stirring extraction time, glue
The surplus ratio of former albumen end peptide is gradually reduced (map drawn after such as Fig. 3 and 5 photodensitometries), this showed with extraction time
Extension, end peptide resection rate be gradually increased.
The discovery of Fig. 2 and 3 is observed, when the digestion time is 32h, a chain N-terminal surplus ratio of collagen is worked as less than 20%
When mixing time is extended for 72h, end peptide is substantially completely cut off；And found from Figure 4 and 5, when the digestion time is 48h, collagen egg
White a chain C-terminal surplus ratio is less than 30%.This shows that method of the invention is suitable for analysis collagen extraction process middle-end peptide and contains
The variation of amount.
The detection of the 3 Ns of end I-type collagen sponge N- antigentic specificity bands of embodiment
Sample description：The collagen protein sponge of each 0.05 ± 0.0005g, tri- batches is weighed respectively, it is each that 10mL sample is added
Buffer solution (100mM trishydroxymethylaminomethane, 8M urea, 2%SDS, 2% mercaptoethanol, pH8.0) mixing, at room temperature, shake
Dissolution 4h is swung, 5min is centrifuged at 5000rmp, takes supernatant spare.
A kind of embodiment of the detection method of collagen peptide end peptide of the invention, includes the following steps：
Sample treatment：3min is handled in 100 DEG C of boiling water baths after the supernatant of 5mL is mixed with 5mL SDS buffer, it is cooling
To room temperature, at 4 DEG C, it is centrifuged 5min under 5000rmp, it is spare to obtain loading sample；Configure 6.5% separation gel and 4% concentration
Glue；Every hole applied sample amount is 10uL, and 30min is run under the electric current of 10mA, after the bromophenol blue in sample enters separation gel, adjustment electricity
Electric current of swimming is 20min until electrophoresis is completed.
Transfer：Nitrocellulose filter, filter paper and the glue of recycling are balanced into 30min in transferring film buffer solution.Transferring film electric current
Control is 4-15 DEG C using ice bath control transferring film temperature, the transferring film time is 90min in 450mA.
Closing and incubation：By transfer film, 2h is closed in concussion at 25 DEG C in 5% skimmed milk power；Followed by ox I type glue
Former n-end of albumen antibody carries out primary antibody incubation to transfer film, and condition is：4 DEG C of incubation 12h；Secondary antibody is with alkali phosphatase enzyme mark
Anti-rabbit IgG is incubated for, and condition is：37 DEG C, 2h.
Chromogenic reaction：Antibody incubation film is placed in chromophoric solution to the 7min that develops the color to occur to band, distilled water terminates reaction.
As a result：If Fig. 6 is shown, the shade of the end the collagen protein sponge N- band of three batches preparation is similar, this table
It is bright when preparing collagen protein sponge, the removing method of collagen end peptide is more stable.
Finally, it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention rather than protects to the present invention
The limitation of range is protected, although the invention is described in detail with reference to the preferred embodiments, those skilled in the art should
Understand, it can be with modification or equivalent replacement of the technical solution of the present invention are made, without departing from the essence of technical solution of the present invention
And range.
Claims (9)
1. a kind of detection method of collagen end peptide, which is characterized in that include the following steps：
(1) sample treatment and electrophoresis：After mixing by sample to be tested and SDS buffer, centrifugation obtains Supernatant samples；Take institute
It states Supernatant samples and carries out SDS-PAGE electrophoresis, recycled after electrophoresis, obtain purpose gel；
(2) albumen transfers：Nitrocellulose filter, filter paper and step (1) resulting gel are balanced 20 in transferring film buffer solution
~30 minutes, transferring film was carried out later, obtains transfer film；
(3) closing and incubation：Transfer film obtained by step (2) is taken, is put into confining liquid and shakes；Then it is anti-that collagen end peptide is added
Body carries out primary antibody incubation to transfer film, and secondary antibody is added later and is incubated for；
(4) chromogenic reaction：Chromophoric solution colour developing is added in transfer film after taking step (3) secondary antibody to be incubated for, until band occurs, then plus
Enter distilled water to react with color development stopping, the type of collagen end peptide in sample to be tested is judged according to gained band.
2. detection method according to claim 1, which is characterized in that the primary antibody be the end ox I-type collagen C- and/or
The end N- antibody.
3. detection method according to claim 1, which is characterized in that the secondary antibody is the anti-rabbit of alkali phosphatase enzyme mark
IgG antibody.
4. detection method according to claim 1, which is characterized in that the sample to be tested is the life of Exogenous Collagen albumen
Object sample or product.
5. detection method according to claim 1, which is characterized in that the detection method further includes step (5)：Using light
Transfer film after chromogenic reaction terminates in density analysis method analytical procedure (4), to judge collagen end peptide in sample to be tested
Content.
6. detection method according to claim 2, which is characterized in that the extension rate of the primary antibody is 1：100~200.
7. detection method according to claim 3, which is characterized in that the extension rate of the secondary antibody is 1：5000~
20000。
8. detection method according to claim 1, which is characterized in that in the step (1), when sample to be tested is to extract
When collagen liquid, can directly it be mixed with SDS buffer；
When sample to be tested is solid, first solid sample is mixed with sample buffer, at room temperature concussion dissolution 4h,
It is centrifuged 5min under 5000rmp, obtains supernatant；
Again by the supernatant or collagen liquid and SDS buffer according to 1:It is handled after the mixing of 1 volume ratio in 100 DEG C of boiling water baths
3~6min after being cooled to room temperature, at 4 DEG C, is centrifuged 5min, obtains Supernatant samples under 5000rmp.
9. detection method according to claim 8, which is characterized in that contain tri- hydroxyl first of 100mM in the sample buffer
Base aminomethane, 8M urea, 2%SDS and 2% mercaptoethanol, the pH value of the sample buffer are 8.0.
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| CN110470846A (en) * | 2019-08-09 | 2019-11-19 | 福建医科大学 | A kind of detection method of Yolk immunoglobulin |
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