CN107179409A - A kind of method extracted based on immunoblotting and show blood latent dactylogram - Google Patents
A kind of method extracted based on immunoblotting and show blood latent dactylogram Download PDFInfo
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- CN107179409A CN107179409A CN201710366131.5A CN201710366131A CN107179409A CN 107179409 A CN107179409 A CN 107179409A CN 201710366131 A CN201710366131 A CN 201710366131A CN 107179409 A CN107179409 A CN 107179409A
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- dactylogram
- latent
- pvdf membrane
- western blot
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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
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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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
Abstract
The invention discloses a kind of method extracted based on immunoblotting and show blood latent dactylogram, belong to fingerprint detection field.The blood latent dactylogram sample remained on different objects is transferred in SDS PAGE glues；Turn mode using wet by target protein and be transferred to pvdf membrane；The primary antibody and the secondary antibody of horseradish peroxidase-labeled of specific recognition target protein are capable of in selection, and using antibody and the specific reaction of target protein, horseradish peroxidase is connected in target detection thing；And then show blood latent dactylogram using chemiluminescence.The present invention can extract the protein component in blood latent dactylogram exactly, while the complete pattern of fingerprint can be retained, i.e., it is difunctional with composition identification and fingerprint manifestation.The present invention has simple, efficient, easy-operating feature, has good application prospect for the blood latent dactylogram that identification criminal case crime scene is left.
Description
First, technical field
The present invention relates to fingerprint detection field, more particularly to one kind is extracted based on Western blot and shows blood latent dactylogram
Method.
2nd, background technology
Fingerprint identification is to carry out one of most reliable method of personal identification, and the current police utilize the case that criminal technique is tracked down
In part, fingerprint recognition accounts for seventy percent or so.Fingerprint is once known as " first of material evidence " by judicial circuit.But as criminal activity is intelligent
The development of degree, the discovery of complete display impression of the hand, recovery rate are decreased obviously.Suspicion human blood has particularly been left on murder scene to refer to
The probability of line is big, but the discovery of blood fingerprint and extraction difficulty are big, and later stage test sensitivity is difficult.
Application publication number discloses " immune based on chemiluminescent enzyme-linked immunosorbent for the Chinese invention patent of " A of CN 103543145 "
The method of the latent fingerprint imaging of analysis ", the antibody-solutions that this method is directly added into finger metabolin on fingerprint are incubated,
It is rinsed after the completion of incubation with buffer solution；Described sample fingerprint is dried, horseradish peroxidase-labeled is then added
Secondary antibody is incubated；After incubation, sample fingerprint is rinsed with cushioning liquid, sample fingerprint is placed in chemiluminescence reaction bottom after drying
In thing solution, fingerprint image is gathered.This method is although simple and quick, but this method is easily by impurity effect (in fingerprint
Grease, dust), the problem of there is non-specific adsorption.
Immunoblotting is one of protein analysis most ripe technology.This method mainly utilizes the Gao Te of antigen-antibody
Different in nature combination principle, by signal amplification, the protein part of substrate is displayed in the form of images, method is directly perceived,
It is convenient.The present invention extracts the protein component in blood latent dactylogram using immunoblotting, can avoid the impurity pair such as grease, dust
The influence of testing result, improves the accuracy of testing result.The present invention provides for the protein component in research blood latent dactylogram
Scientific basis, and the operating efficiency of public security organ's Extraction and discrimination blood latent dactylogram can be effectively improved.
3rd, the content of the invention
In order to make up the deficiencies in the prior art, solve the discovery of blood fingerprint in the prior art and extract difficulty greatly, later stage inspection
The problem of authenticating fixed difficult, the invention provides a kind of method extracted based on Western blot and show blood latent dactylogram.This
Inventive method is simple, the specific good, luminous intensity of detection is high, and fingerprint clear can be shown.
The technical scheme is that：
A kind of method extracted based on Western blot and show blood latent dactylogram, including step：
1) the blood latent dactylogram sample remained on object is transferred on SDS-PAGE glue；
2) destination protein on sample fingerprint is transferred on pvdf membrane；
3) by step 2) 0.5-2h is closed in pvdf membrane immersion skim milk after processing, then clean excessive on pvdf membrane
Milk；
4) to step 3) processing after pvdf membrane on be added dropwise primary antibody solution be incubated, after the completion of incubation clean excessively resist
Body；
5) to step 4) processing after pvdf membrane on be added dropwise horseradish peroxidase-labeled two corresponding anti-solution be incubated, incubate
Multispecific antibody was cleaned after the completion of educating；
6) to step 5) processing after pvdf membrane on be added dropwise in developer solution, camera bellows develop, obtain clearly fingerprint image.
Preferably, step 1) in, the composition of SDS-PAGE glue is as follows：The acryloyl of 4-5mL water, 2-3mL 30%
Amine, 2-3mL concentration be 1.5mol/LpH be 8.8 Tris solution, 0.05-0.15mL 10% SDS solution, 5-7 μ l
TEMED.Protein molecule in fingerprint combines to form negatively charged protein-SDS compounds with SDS, under voltage effect
It is transferred on pvdf membrane.
Preferably, step 2) in destination protein is transferred on pvdf membrane using the wet instrument that turns.
Further, it is wet when turning, voltage 110-130V is set, transfer time is 20-40min.Protein can be made abundant
It is transferred on pvdf membrane.
Preferably, step 3) in skim milk concentration be 4-6% (w/v).The concentration can be effectively combined
Non-specific adsorption sites on pvdf membrane.
Preferably, step 3), 4), 5) in cleaning use TBST buffer solution for cleaning；The TBST buffer solutions are
TBS buffer solutions containing 0.05%Tween20, TBS cushioning liquid is 0.1M Tris.HCl PH7.5,0.15M NaCl.
Preferably, step 4) described in primary antibody solution be rabbit-anti people, the concentration of the primary antibody solution is 0.01-
0.03mg/mL。
Preferably, step 5) described in two corresponding anti-solution be horseradish peroxidase-labeled goat-anti rabbit, the secondary antibody
The concentration of solution is 0.005-0.015mg/mL.
Preferably, step 6) described in developer solution be 0.1M Tris.HCl Shandongs containing 0.4-0.6mM that pH is 8.5
Minot and 0.2-0.3mM are to iodophenol.
The inventive method realizes the highly sensitive detection to blood latent dactylogram, mainly includes：By the latent finger of the blood remained on object
Grain pattern is originally transferred on SDS-PAGE glue；Turn mode using wet by target protein and be transferred to pvdf membrane；Select specific detection mesh
The primary antibody of albumen and the secondary antibody of horseradish peroxidase-labeled are marked, using antibody and the specific reaction of object, by horseradish mistake
Oxide enzyme is connected in target detection thing；Enhanced chemiluminescence substrate produces chemistry hair under horseradish peroxidase enzyme catalytic
Light, so as to show fingerprint and the presence of determinand can be thereby determined that.
Beneficial effects of the present invention are：
1st, the present invention extracts the protein component in blood latent dactylogram, high specificity in advance before addition detection reagent, it is to avoid
Without non-specific adsorption problem；
2nd, the present invention uses pvdf membrane as protein carrier, and the adhesive force of protein is strong；
3rd, method of the invention amplifies protein signal, and sensitivity is high, and luminescence reagent low cost；
4th, the present invention is simple to operate, reliably, stable using traditional protein immunoblot method；
5th, the inventive method is not by impurity effect, and detection confidence is high.
4th, illustrate
In order to illustrate more clearly about the embodiment of the present invention or technical scheme of the prior art, below will be to embodiment or existing
There is the accompanying drawing used required in technology description to be briefly described, it should be apparent that, drawings in the following description are only this
Some embodiments of invention, for those of ordinary skill in the art, without having to pay creative labor, may be used also
To obtain other accompanying drawings according to these accompanying drawings.
Fig. 1 is extracted based on Western blot for the present invention and is shown the method detection blood latent dactylogram signal of blood latent dactylogram
Figure；
Fig. 2 is the hair for the method detection blood latent dactylogram that blood latent dactylogram was extracted and shown to embodiment 1 based on Western blot
Light image.
5th, embodiment
Reagent：
Rabbit anti-human IgG (rabbit-anti people) are purchased from the biological Co., Ltds of Beijing Bo Aosen, article No. bs-0297R；
Goat anti-rabbit IgG/HRP (the goat-anti rabbit of horseradish peroxidase-labeled) are purchased from Beijing Bo Aosensheng
Thing Co., Ltd, article No. bs-0295G-HRP；
Pvdf membrane is purchased from green skies Bioisystech Co., Ltd, article No. FFP24.
Embodiment 1
The method that blood latent dactylogram is extracted and shown based on Western blot, including：
1) bled liquid, be coated onto on finger in finger tip collection one with spring puncture needle, dried；
2) fingerprint is restrained in nonporous glass substrate；Substrate of glass uses absolute ethyl alcohol respectively, and water is cleaned by ultrasonic 20min；
3) sample fingerprint in substrate is transferred on 8% SDS-PAGE glue；
8% SDS-PAGE glue is formulated as follows：
Water：4.6ml；30% acrylamide：2.7ml；1.5M Tris(PH 8.8)：2.5ml；10% SDS：
0.1ml；TEMED：6μl.
4) turn instrument using wet, voltage 120V, transfer time 30min are set, by the purpose of sample fingerprint on SDS-PAGE glue
Protein delivery is on pvdf membrane；
5) after the completion of shifting, pvdf membrane is immersed in 5% (w/v) skim milk and closes 1h；
6) milk excessive on film is cleaned with TBST, is cleaned 3 times, each 5min；
7) to step 6) 0.02mg/ml primary antibodies (rabbit-anti people) are added dropwise on pvdf membrane after processing, 4 DEG C of night incubation primary antibodies are molten
Liquid is incubated；
8) TBST cleans antibody excessive on film, cleans 3 times, each 10min,
9) to step 8) processing after pvdf membrane on be added dropwise horseradish peroxidase-labeled secondary antibody (goat-anti rabbit) normal temperature incubate
1h is educated, secondary antibody concentration is 0.01mg/ml；
10) TBST cleans antibody excessive on film, cleans 3 times, each 10min；
11) (developer solution is 0.1M Tris HCl luminols containing 0.5mM and 0.23mM that PH is 8.5 to iodine to preparing developer liquid
Phenol), it is uniformly dripped into step 10) processing after pvdf membrane on, in camera bellows develop, obtain clear fingerprint image.In horseradish
In the presence of peroxidase, hydrogen peroxide reacts the light for producing that wavelength is 425nm with luminol, so as to show fingerprint.
Wherein, step 6), 8), 10) in TBST be the TBS buffer solutions containing 0.05%Tween-20, TBS cushioning liquid is
0.1M Tris HCl PH7.5、0.15M NaCl。
The present invention is extracted based on Western blot and shows method detection blood latent dactylogram schematic diagram such as Fig. 1 of blood latent dactylogram
It is shown, the primary antibody of specific detection target protein and the secondary antibody of horseradish peroxidase-labeled are selected, antibody and object is utilized
Specific reaction, horseradish peroxidase is connected in target detection thing；Enhanced chemiluminescence substrate is in horseradish peroxidating
Chemiluminescence is produced under thing enzymatic, so as to show fingerprint and the presence of determinand can be thereby determined that.
The present embodiment extracts and shown the illuminated diagram of the method detection blood latent dactylogram of blood latent dactylogram based on Western blot
As shown in Figure 2, it is seen that fingerprint image obtained by the inventive method, fingerprint ridge is very clear.
Embodiment 2
The method that blood latent dactylogram is extracted and shown based on Western blot, including：
1) bled liquid, be coated onto on finger in finger tip collection one with spring puncture needle, dried；
2) fingerprint is restrained in porous paper substrates；Paper substrates are common A4 paper；
3) sample fingerprint in substrate is transferred on 10% SDS-PAGE glue；
10% SDS-PAGE glue is formulated as follows：
Water：4.1ml；30% acrylamide：3.3ml；1.5M Tris(PH 8.8)：2.5ml；10% SDS：
0.1ml；TEMED：6μl.
4) turn instrument using wet, voltage 110V, transfer time 30min are set, by the purpose of sample fingerprint on SDS-PAGE glue
Protein delivery is on pvdf membrane；
5) after the completion of shifting, pvdf membrane is immersed in 4% (w/v) skim milk and closes 1.5h；
6) milk excessive on film is cleaned with TBST, is cleaned 3 times, each 5min；
7) to step 6) 0.03mg/ml primary antibodies (rabbit-anti people) are added dropwise on pvdf membrane after processing, 4 DEG C of night incubation primary antibodies are molten
Liquid is incubated；
8) TBST cleans antibody excessive on film, cleans 3 times, each 10min,
9) to step 8) processing after pvdf membrane on be added dropwise horseradish peroxidase-labeled secondary antibody (goat-anti rabbit) normal temperature incubate
1h is educated, secondary antibody concentration is 0.02mg/ml；
10) TBST cleans antibody excessive on film, cleans 3 times, each 10min；
11) (developer solution is 0.1M Tris HCl luminols containing 0.5mM and 0.23mM that PH is 8.5 to iodine to preparing developer liquid
Phenol), it is uniformly dripped into step 10) processing after pvdf membrane on, in camera bellows develop, obtain clear fingerprint image.In horseradish
In the presence of peroxidase, hydrogen peroxide reacts the light for producing that wavelength is 425nm with luminol, so as to show fingerprint.
Wherein, step 6), 8), 10) in TBST be the TBS buffer solutions containing 0.05%Tween-20, TBS cushioning liquid is
0.1M TrisHCl PH7.5、0.15M NaCl。
Claims (9)
1. a kind of method extracted based on Western blot and show blood latent dactylogram, including step：
1) the blood latent dactylogram sample remained on object is transferred on SDS-PAGE glue；
2) destination protein on finger mark sample is transferred on pvdf membrane；
3) by step 2) 0.5-2h is closed in pvdf membrane immersion skim milk after processing, then clean excessive ox on pvdf membrane
Milk；
4) to step 3) processing after pvdf membrane on be added dropwise primary antibody solution be incubated, multispecific antibody was cleaned after the completion of incubation；
5) to step 4) processing after pvdf membrane on be added dropwise horseradish peroxidase-labeled two corresponding anti-solution be incubated, be incubated
Multispecific antibody was cleaned after；
6) to step 5) processing after pvdf membrane on be added dropwise in developer solution, camera bellows develop, obtain clearly fingerprint image.
2. the method that blood latent dactylogram is extracted and shown based on Western blot as claimed in claim 1, it is characterised in that step
1) in, the composition of SDS-PAGE glue is as follows：4-5mL water, 2-3mL 30% acrylamide, 2-3mL concentration are 1.5mol/L pH
For 8.8 Tris solution, 0.05-0.15mL 10% SDS solution, 5-7 lTEMED.
3. the method that blood latent dactylogram is extracted and shown based on Western blot as claimed in claim 1 or 2, it is characterised in that：
Step 2) in destination protein is transferred on pvdf membrane using the wet instrument that turns.
4. the method that blood latent dactylogram is extracted and shown based on Western blot as claimed in claim 3, it is characterised in that：Wet turn
When, voltage 110-130V is set, transfer time is 20-40min.
5. the method that blood latent dactylogram is extracted and shown based on Western blot as claimed in claim 1 or 2, it is characterised in that：
Step 3) in skim milk concentration be 4-6%.
6. the method that blood latent dactylogram is extracted and shown based on Western blot as claimed in claim 1, it is characterised in that：Step
3), 4), 5) middle clean uses TBST buffer solution for cleaning；The TBST buffer solutions are the TBS bufferings containing 0.05%Tween20
Liquid, TBS cushioning liquid is 0.1M Tris HCl PH7.5,0.15M NaCl.
7. the method that blood latent dactylogram is extracted and shown based on Western blot as claimed in claim 1, it is characterised in that：Step
4) primary antibody solution described in is rabbit-anti people, and the concentration of the primary antibody solution is 0.01-0.03mg/mL.
8. the method that blood latent dactylogram is extracted and shown based on Western blot as claimed in claim 1, it is characterised in that：Step
5) two corresponding anti-solution described in is horseradish peroxidase-labeled goat-anti rabbit, and the concentration of the two corresponding anti-solution is 0.005-0.015mg/
mL。
9. the method that blood latent dactylogram is extracted and shown based on Western blot as claimed in claim 1, it is characterised in that：Step
6) developer solution described in is 0.1M Tris HCl luminols containing 0.4-0.6mM and 0.2-0.3mM that pH is 8.5 to iodophenol.
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Inventor after: Xu Linru Inventor after: Liu Ehu Inventor after: Lv Zhimin Inventor before: Liu Ehu Inventor before: Xu Linru Inventor before: Lv Zhimin |