CN109444116A - A kind of enhanced ECL chemical illuminating reagent - Google Patents
A kind of enhanced ECL chemical illuminating reagent Download PDFInfo
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- CN109444116A CN109444116A CN201811209166.9A CN201811209166A CN109444116A CN 109444116 A CN109444116 A CN 109444116A CN 201811209166 A CN201811209166 A CN 201811209166A CN 109444116 A CN109444116 A CN 109444116A
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- chemical illuminating
- illuminating reagent
- luminol
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- 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
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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
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Abstract
The invention discloses a kind of enhanced ECL chemical illuminating reagents, the reagent has the formula of optimization, it can be matched in Western Blot and ELISA experiment with horseradish peroxidase (HRP), compatible exposure and CCD scanning, high sensitivity can detect the antigen of pg rank, and luminous intensity can be enhanced, anti- quenching ability is strong, as a result stable, and imaging effect is obvious.
Description
Technical field
The invention belongs to technical field of immunoassay, are related to a kind of enhanced ECL chemical illuminating reagent.
Background technique
Whether a certain protein expression level changes in research body tissue or cell, almost requires using albumen
Matter immunoblotting (Western Blot) method detects, and immunoblotting is finally also that a most important step is exactly enzyme labelled antibody and bottom
Object reacts release signal.Wherein relatively simple is also substrate stoichiometric for everybody generally accepted Western blot coloration method
Luminescence method ECL.
Common ECL series of products in Western Blot are for detecting horseradish peroxidase during immunoblotting
The chemiluminescent substrate of enzyme (HRP) labelled antibody.HRP enzyme system has high conversion coefficient, has to different sensitivity analysis system
There is versatility and is widely applied.Currently, the most common luminous substrate of HRP enzyme system is luminol, different luminol and its spreads out
Two kinds of isometric solution are mixed, are incubated for the film of trace by biology, and film or imaging system then can be used
System testing result.Its basic detection process includes: that protein or nucleic acid are transferred on blotting membrane after electrophoresis, with horseradish peroxidating
Destination protein on the antibody combination film of object enzyme HRP label, or the core on film is combined directly or indirectly with the probe that HRP is marked
Acid is used the ECL chemiluminescence working solution prepared after washing film, is incubated at room temperature under visible light several minutes, by blotting membrane preservative film
Coating is fixed on X-ray exposure magazine, is then transferred to darkroom for film and is pressed in exposure several seconds to a few hours on film.After developing fixing
Protein or nucleic acid bands can be clearly shown on X-ray.CCD directly can also be carried out to blotting membrane and swept without exposure
It retouches.
However, the shining as flash type of Luminol, intensity is weak, the duration is short, luminous efficiency is not high.Using Shandong
The chemiluminescence immunoassay detection of Dumio system label usually faces following problems: 1, western development effect is bad；2, strong light
It spends not high；3, easy to form to be quenched.
Summary of the invention
To solve the above-mentioned problems, the present invention provides a kind of enhanced ECL chemical illuminating reagent of high sensitivity, the examinations
Agent can match in Western Blot and ELISA experiment with horseradish peroxidase (HRP), and luminous intensity is high, development
Effect is good and is not easy to be quenched, and can detect the antigen of pg rank.
To achieve the goals above, the invention adopts the following technical scheme:
A kind of enhanced ECL chemical illuminating reagent, including component 1 and component 2, in which:
Component 1 includes: again
Component 2 includes: again
Tris-HCl buffer, pH 8.0-9.0 0.1-0.3M
30%-50% dioxygen water volume fraction 0.04%-3%.
Preferably, the Derivative of Luminol is luminol sodium salt, different luminol or 4- the amino different Shandong of base-N- ethyl
Promise.
Preferably, in component 1 and component 2, the pH of Tris-HCl buffer is 9.0.
Preferably, the volume ratio of component 1 and component 2 is 1:1-2:1.
Preferably, in component 1, the luminol is to be dissolved in the luminol mass concentration that dimethyl sulfoxide is formulated to be
The luminol solution of 60-120mg/ml, the p-Coumaric Acid be use pH for 9.0 0.1M Tris-HCl buffer and
At p-Coumaric Acid molar concentration be 5-8mM p-Coumaric Acid solution.
Preferably, component 1 includes:
Component 2 includes:
0.1-0.3M Tris-HCl buffer, 9.0 20-200ml of pH
30%-50% hydrogen peroxide 0.1-0.6ml.
Preferably, the volume ratio of component 1 and component 2 is 2:1.
The present invention provides a kind of western developing agents of high quality, have the following beneficial effects:
1) formula optimized enhances luminous intensity；
2) anti-quenching ability is strong；
3) compatible exposure and CCD scanning；
4) result is stablized, and imaging effect is obvious.
Detailed description of the invention
Fig. 1 is experiment flow figure of the invention.
Fig. 2 is the present invention and prior art development effect comparative diagram.
Specific embodiment
The present invention is further illustrated with embodiment below, but following embodiment is not construed as to the scope of the present invention
It limits.Material therefor and reagent are commercial product in embodiment, do not indicate the experimental method of actual conditions further, usually press
More solito condition, or according to the normal condition proposed by manufacturer.Heretofore described " room temperature " refers to the operation tested
Between temperature, generally 25 DEG C.
The preparation of the enhanced ECL chemical illuminating reagent of embodiment 1
Component 1 and component 2 are used in mixed way according to 2:1, ready-to-use.
The preparation of the enhanced ECL chemical illuminating reagent of embodiment 2
Component 1 and component 2 are used in mixed way according to 2:1, ready-to-use.
The preparation of the enhanced ECL chemical illuminating reagent of embodiment 3
Component 1 and component 2 are used in mixed way according to 1:1, ready-to-use.
As described in summary of the invention, in above 3 embodiments, the luminol is dissolved in dimethyl sulfoxide and is formulated
Solution, the p-Coumaric Acid are that pH is used to form solution for 9.0 0.1M Tris-HCl buffer.
The preparation of the enhanced ECL chemical illuminating reagent of embodiment 4
Component 1 and component 2 are used in mixed way according to 2:1, ready-to-use.
Experimental example
One, experimental material
1, cell line and source:
Ovarian epithelial carcinoma Cell line SKOV3, is purchased from Shanghai Cell Bank of the Chinese Academy of Sciences.It is based on Gibco cell culture
37 DEG C, 5%CO2Under the conditions of cultivate.
2, reagent:
RIPA cell pyrolysis liquid (C1053, Beijing Puli's lema gene Technology Co., Ltd.)
BCA protein quantification kit (BCA Protein Assay Kit) (CW0014S, health are century)
Acrylamide (Acrylamide) (J173005, Aladdin)
Bisacrylamide (Bis-acrylamide) (Tianjin great Mao chemical reagent factory)
Trishydroxymethylaminomethane (Tris) (T8060, Solarbio)
Lauryl sodium sulfate (SDS) (151-21-3, western Gansu Province science limited liability company)
Ammonium persulfate (Sodium persulfate) (Tianjin Zhi Yuan chemical reagent Co., Ltd)
Glycine (Glycine) (G8200, Solarbio)
Sodium chloride (NaCl) (7647-14-5, Tianjin great Mao chemical reagent factory)
Potassium chloride (KCl) (Tianjin Zhi Yuan chemical reagent Co., Ltd)
Tetramethylethylenediamine (TEMED) (T105496, Aladdin)
Marker (#26617, Thermo)
Pvdf membrane (IPVH00010, Millipore)
Close dedicated skimmed milk power (P1622, Beijing Puli's lema gene Technology Co., Ltd.)
Bovine serum albumin(BSA) (BSA) (A8020, Suo Laibao)
Super quick luminescent solution (RJ239676 matches silent fly)
Internal reference primary antibody: Mouse Monoclonal Anti-GAPDH (TA-08, Zhong Shan Golden Bridge, 1/2000)
Secondary antibody: horseradish enzyme marks mountain sheep anti-mouse igg (H+L) (ZB-2305, Zhong Shan Golden Bridge, 1/5000)
Luminol (Sigma A8511)
P-Coumaric Acid (Sigma C9008)
1,4- diazabicylo [2.2.2] octane (Sigma D2522)
3, instrument and equipment:
Single track adjustable pipette (0.5-10 μ l, Mettler-Toledo Instrument (Shanghai) Co., Ltd.)
Single track adjustable pipette (20-200 μ l, Mettler-Toledo Instrument (Shanghai) Co., Ltd.)
Single track adjustable pipette (100-1000 μ l, Mettler-Toledo Instrument (Shanghai) Co., Ltd.)
Freezing high speed is from (TGL-16D, Changzhou Zhong Jie laboratory apparatus Manufacturing Co., Ltd)
Digital display thermostat water bath (HH-2, the loud and clear experimental instruments and equipment limited in Zhengzhou)
Ultraviolet specrophotometer (UV-1600PC, Shanghai U.S. spectrum reach Instrument Ltd.)
Albumen vertical electrophoresis apparatus (DYY-6C, Beijing Liuyi Instrument Factory)
Constant-temperature table (TC-100B, Shanghai neck is at Biotechnology Co., Ltd)
(Chemi DocTM XRS+, Bole's life medical product (Shanghai) have hypersensitivity chemiluminescence imaging system
Limit company)
Two, experimental method
Experiment flow of the invention is as shown in Figure 1, specifically include that
Albumen → BCA determination of protein concentration → glue → loading → electrophoresis → transferring film → closing → incubation primary antibody → is extracted to incubate
Educate secondary antibody → exposure development.
Specific experimental method is as follows:
1, Western Blot main agents are prepared:
30% acrylamide: acrylamide (Acrylamide) 72.5g, bisacrylamide 2.5g adds water to be settled to
250mL。
Tris 8.8:Tris 27.225g, pH are adjusted to 8.8, and water is added to be settled to 150mL.
Tris 6.8:Tris 6.05g, pH are adjusted to 6.8, and water is added to be settled to 50mL.
10%SDS:SDS 10g, adds water to be settled to 100mL.
APS: ammonium persulfate 1g adds water to be settled to 10mL.
10 × electrophoresis liquid: Tris 30.2g, glycine 188g, SDS 10g, pH are adjusted to 8.3, and water is added to be settled to 1000mL.
10 × transferring film liquid: Tris 58g, glycine 29g, SDS 3.75g, pH are adjusted to 8.3, and water is added to be settled to 1000mL.
10 × TBST:NaCl 80g, Tris 30g, KCl 2g, pH are adjusted to 7.4, and water is added to be settled to 1000mL.
1 × electrophoresis liquid: 10 × electrophoresis liquid of 100mL adds water to 1000mL.
1 × transferring film liquid: 10 × transferring film of 100mL liquid+200mL methanol plus water to 1000mL.
1 × TBST:100mL, 10 × TBS, adds water to 1000mL, adds tween 1mL.
2, SKOV3 cell protein extracts
(1) cell culture fluid in culture dish is discarded.
(2) cell pyrolysis liquid of 100 μ l is added in every hole, is placed in 15min on ice.
(3) cell is blown and beaten with pipette tips, is put into the EP pipe marked.
(4) 12000r/min supercentrifuge is centrifuged 10min.Supernatant is taken, new EP pipe (BCA measurement) is moved to, it is heavy to abandon
It forms sediment.
(5) 1 × PBS buffer solution (pH=7.4) is added, boiling water boiling 5min.
(6) -20 DEG C of preservations are placed in, it is spare as testing protein sample.
3, protein concentration is measured:
(1) BSA standard items are diluted:
BSA standard items are diluted with the dilution according to the form below consistent with sample to be tested.
Note: dilution is with 1 × PBS phosphate buffer (pH=6-8).
(2) BCA working solution is prepared:
1) BCA working solution total amount is calculated
BCA working solution total amount=(BSA standard items number of samples+unknown sample number) × multiple holes number × each sample B CA
Working solution volume
Note:, generally can 1~2 hole of polygamy system when preparing BCA working solution since there may be errors for sample-adding.
2) total amount is needed according to calculated BCA working solution, by BCA-A and BCA-B according to the volume ratio of 50:1, prepared
BCA working solution, mixes well.
(3) quantitative detection (using test tube detection method)
1) the A-G BSA standard items diluted and each 5 μ l of testing protein sample are added separately in the test tube marked.
2) 100 μ l BCA working solutions are respectively added into each test tube, mixes well, is incubated for 30min in 37 DEG C of water-baths, it will be each
Pipe is cooled to room temperature, and detection must be completed in 3-5min.
3) its data is measured at OD 562 with ultraviolet specrophotometer, measure the extinction of BSA standard items and each sample
Value, makes a record simultaneously.
4) standard curve is drawn, the protein concentration in sample is calculated.
| Sample ID | Absorbance (Abs) | Protein concentration (μ g/ μ l) |
| SKOV3 cell | 1.979 | 3.87547095 |
4, protein electrophoresis extremely exposes:
(1) SDS-PAGE glue is prepared
Note: the 10% separation gel volume this time prepared is 10ml.
Note: the 5% concentrating colloidal product this time prepared is 10ml.
(2) separation gel that will be prepared in (1) is slowly added in the plastic plate that frame is good, until 2/3 position of plate.Add
Enter dehydrated alcohol moulding.After gelling to be separated is solid, concentration glue is added, and plug stripping fork.
(3) after concentration glue solidifies completely, 1 × electrophoresis liquid is prepared.
(4) shelf is put into 1 × electrophoresis liquid and takes out stripping fork, the protein sample and Marker of certain volume is added.
Albumen applied sample amount:
| Sample ID | Applied sample amount (μ l) |
| SKOV3 cell | 10 |
(5) 1 × electrophoresis liquid is prepared, offset plate is submerged with 1 × electrophoresis liquid, starts electrophoresis, 60V compression protein, 80V protein isolate
(120min)。
(6) 1 × transferring film liquid is prepared when band is run to offset plate half, and be pre-chilled.
(7) position of band is cut pvdf membrane (about 1-2CM) on the estimation, and methanol activates 15s.
(8) the size properly glue containing internal reference or purpose band is cut.
(9) " sandwich " (sponge-filter paper-glue-film-filter paper-sponge) is put well.
(10) " sandwich " is submerged with 1 × transferring film liquid, with 4 DEG C of transferring films of 300mA constant current 20 minutes 1 hour.
| Index name | Molecular weight | Electrophoresis (60V) time | Electrophoresis (80V) time | Transferring film (300mA) time |
| GAPDH | 36KD | 30min | 2h | 1h20min |
Note: electrophoresis and the transferring film time that this table is index GAPDH
(11) with the skim milk confining liquid of 1 × TBST preparation 3%, (6g closes dedicated skimmed milk power, and 1 × TBST is diluted to
200ml), closing is stayed overnight.
(12) 1 anti-dilution (according to antibody specification recommended density, 1:2000 dilution) is prepared, pvdf membrane is incubated for primary antibody
3 hours.
(13) film is washed, 1 × TBST is discarded after impregnating 10min with 1 × TBST, is repeated 3 times.
(14) 2 anti-dilutions (according to antibody specification recommended density, 1:5000 dilution) are prepared, pvdf membrane is incubated for secondary antibody
2 hours.
(15) film is washed, 1 × TBST is discarded after impregnating 10min with 1 × TBST, is repeated 3 times.
(16) the enhanced ECL chemical illuminating reagent for preparing embodiment 2 is used for experimental example, super quick by what is bought on the market
Luminescent solution (RJ239676 matches silent fly) is used for comparative example (prior art is shown as in Fig. 2), soaks PVDF with two kinds of luminescent solutions respectively
Operation program development imaging in hypersensitivity chemiluminescence imaging system sample rest area is placed in after film.Above-mentioned experimental example with it is right
Ratio is compared, and in addition to the luminescence reagent of use is different, other experiment conditions are identical.
As shown in Fig. 2, comparing with super quick luminescent solution (RJ239676 matches silent fly) disclosed in the prior art, the present invention is mentioned
The enhanced ECL chemical illuminating reagent supplied effectively enhances luminol-hydrogen peroxide chemistry after optimizing to existing formula
The luminous intensity of luminescence system, development imaging effect is obvious, while the chemiluminescence signal generated can be protected in a long time
It is fixed to keep steady, and is not easy to be quenched.
Claims (7)
1. a kind of enhanced ECL chemical illuminating reagent, including component 1 and component 2, in which:
Component 1 includes: again
Component 2 includes: again
Tris-HCl buffer, pH 8.0-9.0 0.1-0.3M
30%-50% dioxygen water volume fraction 0.04%-3%.
2. a kind of enhanced ECL chemical illuminating reagent according to claim 1, which is characterized in that the luminol is derivative
Object is luminol sodium salt, different luminol or 4- the amino different Lu Nuo of base-N- ethyl.
3. a kind of enhanced ECL chemical illuminating reagent according to claim 1, which is characterized in that in component 1 and component 2,
The pH of Tris-HCl buffer is 9.0.
4. a kind of enhanced ECL chemical illuminating reagent according to claim 1, which is characterized in that component 1 and component 2
Volume ratio is 1:1-2:1.
5. a kind of enhanced ECL chemical illuminating reagent according to claim 1, which is characterized in that in component 1, the Shandong
Minot is that be dissolved in the luminol mass concentration that dimethyl sulfoxide is formulated be 60-
The luminol solution of 120mg/ml, the p-Coumaric Acid be use pH for 9.0 0.1M Tris-HCl buffer and
At p-Coumaric Acid molar concentration be 5-8mM p-Coumaric Acid solution.
6. a kind of enhanced ECL chemical illuminating reagent according to claim 5, which is characterized in that component 1 includes:
Component 2 includes:
0.1-0.3M Tris-HCl buffer, 9.0 20-200ml of pH
30%-50% hydrogen peroxide 0.1-0.6ml.
7. a kind of enhanced ECL chemical illuminating reagent according to claim 6, which is characterized in that component 1 and component 2
Volume ratio is 2:1.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201811209166.9A CN109444116A (en) | 2018-10-17 | 2018-10-17 | A kind of enhanced ECL chemical illuminating reagent |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811209166.9A CN109444116A (en) | 2018-10-17 | 2018-10-17 | A kind of enhanced ECL chemical illuminating reagent |
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| CN109444116A true CN109444116A (en) | 2019-03-08 |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110554183A (en) * | 2019-09-17 | 2019-12-10 | 沈阳万类生物科技有限公司 | Hypersensitive ECL chemiluminescent reagent |
| US20220373554A1 (en) * | 2019-01-03 | 2022-11-24 | Meso Scale Technologies, Llc. | Compositions and methods for carrying out assay measurements |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5380650A (en) * | 1986-09-03 | 1995-01-10 | British Technology Group Ltd. | Enhanced luminescent assay |
| CN101281197A (en) * | 2008-05-16 | 2008-10-08 | 北京工业大学 | HIV-1 p24 antigen luminol chemiluminescence immune analyse detecting method |
| CN102313732B (en) * | 2010-07-06 | 2017-10-17 | 喜雅纳肯有限公司 | Enhancing and the photoemissive method of regulation chemiluminescence reaction |
-
2018
- 2018-10-17 CN CN201811209166.9A patent/CN109444116A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5380650A (en) * | 1986-09-03 | 1995-01-10 | British Technology Group Ltd. | Enhanced luminescent assay |
| CN101281197A (en) * | 2008-05-16 | 2008-10-08 | 北京工业大学 | HIV-1 p24 antigen luminol chemiluminescence immune analyse detecting method |
| CN102313732B (en) * | 2010-07-06 | 2017-10-17 | 喜雅纳肯有限公司 | Enhancing and the photoemissive method of regulation chemiluminescence reaction |
Non-Patent Citations (1)
| Title |
|---|
| DOLORES D. MRUK ET AL.,: ""Enhanced chemiluminescence (ECL) for routine immunoblotting"", 《SPERMATOGENESIS》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20220373554A1 (en) * | 2019-01-03 | 2022-11-24 | Meso Scale Technologies, Llc. | Compositions and methods for carrying out assay measurements |
| US11927592B2 (en) * | 2019-01-03 | 2024-03-12 | Meso Scale Technologies, Llc. | Compositions and methods for carrying out assay measurements |
| CN110554183A (en) * | 2019-09-17 | 2019-12-10 | 沈阳万类生物科技有限公司 | Hypersensitive ECL chemiluminescent reagent |
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