CN101846625B - Method for measuring concentration of protein in solution by using glutaraldehyde to induce infrared fluorescence - Google Patents
Method for measuring concentration of protein in solution by using glutaraldehyde to induce infrared fluorescence Download PDFInfo
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- CN101846625B CN101846625B CN2010101714903A CN201010171490A CN101846625B CN 101846625 B CN101846625 B CN 101846625B CN 2010101714903 A CN2010101714903 A CN 2010101714903A CN 201010171490 A CN201010171490 A CN 201010171490A CN 101846625 B CN101846625 B CN 101846625B
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Abstract
The invention discloses a method for measuring concentration of protein in solution by using glutaraldehyde to induce infrared fluorescence. The method comprises the following steps: dropwise adding protein solution in an experimental group to be tested and a plurality of protein solution with known concentration in a criterion group on nitrocellulose membrane; after drying, placing the obtained mixture into glutaraldehyde solution, and then washing after heating; and detecting fluorescence intensity of samples, and calculating the protein concentration in the experimental group according to a standard curve drawn by the known protein concentration and the fluorescence intensity and the fluorescence intensity of the protein samples in the experimental group. The method has lowered cost, improved speed and reduced operating time, thereby facilitating bulk operation.
Description
Technical field
The present invention relates to a kind of measuring method, especially a kind of method of measuring protein concentration.
Background technology
Determination of protein concentration is the technology of in life science, often using.Have many methods to be used for determination of protein concentration at present, but limitation is respectively arranged again: classical ultraviolet spectrophotometry sensitivity is limited; Conventional BCA, Lowry method etc. relate to the reagent preparation of relative complex again; The method that has is the Chinese patent operation steps complicacy of CN1687745 like publication number, introduces personal error easily, causes the accuracy of measurement result to descend; The method that has is like U.S. Pat 2010047913 etc., with LC and mass spectrometry; Though improved the sensitivity and the accuracy that detect; But because the costliness of chromatography and mass spectroscopy device has limited its use, and these class methods are consuming time longer, are not easy to realize batch operation fast.
Summary of the invention
Deficiencies such as the prior art counting is with high costs in order to overcome, operation length consuming time the invention provides a kind of method of measuring protein concentration, and are with low cost, simple and efficient to handle.
The technical solution adopted for the present invention to solve the technical problems may further comprise the steps:
1. each 0.5-2 μ L of the standard group protein solution that the experimental group protein solution of unknown concentration to be measured is different with 1 group of at least 4 concentration known and concentration drips on nitrocellulose filter;
2. nitrocellulose filter is dried naturally;
3. be 0.1%～10% glutaraldehyde solution with the whole mass percent concentration of putting into of nitrocellulose filter, solution and nitrocellulose filter be heated to 40 ℃～80 ℃, and be incubated 5～60 minutes;
4. clean nitrocellulose filter more than twice with phosphate buffer (PBS) or water or physiological saline;
5. use the infrared laser scanning imaging system, select that arbitrary wavelength laser excites between the 660nm-710nm, greater than the excitation laser wavelength fluorescence intensity of each protein solution spot on the 40nm place detection nitrocellulose filter at least;
6. the known protein matter concentration of combined standard histone matter solution and fluorescence intensity drawing standard curve obtain fluorescence intensity I
StandardWith protein solution concentration N
StandardRelation formula: I
Standard=a * N
Standard+ b, wherein a is the monobasic linearly dependent coefficient, b is an intercept, all calculates through typical curve and learns;
7. according to the fluorescence intensity I of typical curve and experimental group protein solution spot
ExperimentExperiment with computing histone matter solution concentration N
Experiment, i.e. N
Experiment=(I
Experiment-b)/a.
Though because experimental group protein solution unknown concentration generally to be measured; But the order of magnitude of its concentration is confirmable basically; Therefore; The protein concentration that should have one in the said standard group protein solution at least is greater than experimental group protein solution concentration, and has one protein concentration at least less than experimental group protein solution concentration.Otherwise will cause to obtain described typical curve, just need readjust standard group protein solution concentration.
Said nitrocellulose filter should be enough big, makes not overlapped interference between the spot that forms after each protein solution dropping above that at least.
Described glutaraldehyde solution adopts water or physiological saline or phosphate buffer (PBS) to prepare as solvent.
The invention has the beneficial effects as follows:, when playing the fixing protein effect, utilize glutaraldehyde and protein interaction to produce this phenomenon of IR fluorescence owing to adopt glutaraldehyde solution to have the nitrocellulose filter of protein example to carry out immersion treatment to point; In conjunction with the IR fluorescence checkout equipment protein is carried out quantitatively; Compared with prior art, the present invention has embodied and has reduced cost, raising speed; Reduce the running time, be beneficial to batch operation.
Below in conjunction with embodiment the present invention is further specified.
Embodiment
Embodiment 1: use the method for the invention to carry out quantification of protein
1. bovine serum albumin(BSA) (BSA) is with unknown concentration (experimental group) and concentration known (standard group) 1250ng/ μ L, 625ng/ μ L, 312.5ng/ μ L, 156.25ng/ μ L, 78.125ng/ μ L, 39.06ng/ μ L, 19.5ng/ μ L, 9.75ng/ μ L, 4.88ng/ μ L solution, and each sample 2 μ L drips on nitrocellulose filter;
2. nitrocellulose filter is dried naturally;
3. be 0.1% normal saline solution with the whole glutaraldehyde concentration of putting into of nitrocellulose filter, solution is heated to 80 ℃, be incubated 30 minutes;
4. water cleans nitrocellulose filter more than twice;
5. use the infrared laser scanning imaging system, select the 680nm wavelength laser to excite, in fluorescence intensity greater than excitation laser wavelength 720nm place test sample;
6. combined standard group known protein matter concentration and fluorescence intensity drawing standard curve obtain fluorescence intensity I
StandardWith protein concentration N
StandardRelation formula: I
Standard=a * N
Standard+ b, wherein a is the monobasic linearly dependent coefficient, b is an intercept, all calculates through typical curve and learns;
7. according to the fluorescence intensity (I of typical curve and experimental group protein example
Experiment) experiment with computing histone matter concentration (N
Experiment), i.e. N
Experiment=(I
Experiment-b)/a.
Adopt method pair cell of the present invention to count, wide in the range of linearity, the data result collimation is good, the high (R of linear
2=0.998), the 720nm place fluorescence intensity according to gained equation with one unknown quantity and measurement sample can calculate the protein concentration in the sample fast and accurately.
Embodiment 2: use the method for the invention to carry out quantification of protein
1. bovine serum albumin(BSA) (BSA) is with unknown concentration (experimental group) and concentration known (standard group) 625ng/ μ L, 312.5ng/ μ L, 156.25ng/ μ L, 78.13ng/ μ L, 39.06ng/ μ L, 19.5ng/ μ L, 9.75ng/ μ L, 4.88ng/ μ L, 2.44ng/ μ L solution, and each sample 1 μ L drips on nitrocellulose filter;
2. nitrocellulose filter is dried naturally;
3. be 1% phosphate buffer (PBS) solution with the whole glutaraldehyde concentration of putting into of nitrocellulose filter, solution is placed the micro-wave oven heating, reacted 5 minutes;
4. clean nitrocellulose filter more than twice with phosphate buffer;
5. use the infrared laser scanning imaging system, select the 710nm wavelength laser to excite, in fluorescence intensity greater than excitation laser wavelength 780nm place test sample;
6. combined standard group known protein matter concentration and fluorescence intensity drawing standard curve obtain fluorescence intensity I
StandardWith protein concentration N
StandardRelation formula: I
Standard=a * N
Standard+ b, wherein a is the monobasic linearly dependent coefficient, b is an intercept, all calculates through typical curve and learns;
7. according to the fluorescence intensity (I of typical curve and experimental group protein example
Experiment) experiment with computing histone matter concentration (N
Experiment), i.e. N
Experiment=(I
Experiment-b)/a.
Adopt method pair cell of the present invention to count, wide in the range of linearity, the data result collimation is good, the high (R of linear
2=0.997), the 780nm place fluorescence intensity according to gained equation with one unknown quantity and measurement sample can calculate the protein concentration in the sample fast and accurately.
Embodiment 3: use the method for the invention to carry out quantification of protein
1. bovine serum albumin(BSA) (BSA) is with unknown concentration (experimental group) and concentration known (standard group) 2500ng/ μ L, 1250ng/ μ L, 625ng/ μ L, 312.5ng/ μ L, 156.25ng/ μ L, 78.125ng/ μ L, 39.06ng/ μ L, 19.5ng/ μ L, 9.75ng/ μ L, solution, and each sample 0.5 μ L drips on nitrocellulose filter;
2. nitrocellulose filter is dried naturally;
3. be 10% the WS with the whole glutaraldehyde concentration of putting into of nitrocellulose filter, solution is heated to 40 ℃, be incubated 60 minutes;
4. clean nitrocellulose filter more than twice with physiological saline;
5. use the infrared laser scanning imaging system, select the 690nm wavelength laser to excite, in fluorescence intensity greater than excitation laser wavelength 740nm place test sample;
6. combined standard group known protein matter concentration and fluorescence intensity drawing standard curve obtain fluorescence intensity I
StandardWith protein concentration N
StandardRelation formula: I
Standard=a * N
Standard+ b, wherein a is the monobasic linearly dependent coefficient, b is an intercept, all calculates through typical curve and learns;
7. according to the fluorescence intensity (I of typical curve and experimental group protein example
Experiment) experiment with computing histone matter concentration (N
Experiment), i.e. N
Experiment=(I
Experiment-b)/a.
Adopt method pair cell of the present invention to count, wide in the range of linearity, the data result collimation is good, the high (R of linear
2=0.998), the 740nm place fluorescence intensity according to gained equation with one unknown quantity and measurement sample can calculate the protein concentration in the sample fast and accurately.
Claims (4)
1. utilize glutaraldehyde to bring out the method that IR fluorescence is measured concentration of protein in solution, it is characterized in that comprising the steps:
1) the experimental group protein solution of unknown concentration to be measured is different with 1 group of at least 4 concentration known and concentration each 0.5-2 μ L of standard group protein solution drip on nitrocellulose filter;
2) nitrocellulose filter is dried naturally;
3) be 0.1%～10% glutaraldehyde solution with the whole mass percent concentration of putting into of nitrocellulose filter, solution and nitrocellulose filter be heated to 40 ℃～80 ℃, and be incubated 5～60 minutes;
4) clean nitrocellulose filter more than twice with phosphate buffer or water or physiological saline;
5) use the infrared laser scanning imaging system, select that arbitrary wavelength laser excites between the 660nm-710nm, greater than the excitation laser wavelength fluorescence intensity of each protein solution spot on the 40nm place detection nitrocellulose filter at least;
6) combined standard group known protein matter concentration and fluorescence intensity drawing standard curve obtain fluorescence intensity I
StandardWith protein solution concentration N
StandardRelation formula: I
Standard=a * N
Standard+ b, wherein a is the monobasic linearly dependent coefficient, b is an intercept, all calculates through typical curve and learns;
7) according to the fluorescence intensity I of typical curve and experimental group protein solution spot
ExperimentExperiment with computing histone matter solution concentration N
Experiment, i.e. N
Experiment=(I
Experiment-b)/a.
2. the glutaraldehyde that utilizes according to claim 1 brings out the method that IR fluorescence is measured concentration of protein in solution; It is characterized in that: the protein concentration that should have in the described standard group protein solution at least is greater than experimental group protein solution concentration, and has one protein concentration at least less than experimental group protein solution concentration.
3. the glutaraldehyde that utilizes according to claim 1 brings out the method that IR fluorescence is measured concentration of protein in solution, it is characterized in that: not overlapped interference is as the criterion described nitrocellulose filter size between the spot that back above that forms so that each protein solution drips.
4. the glutaraldehyde that utilizes according to claim 1 brings out the method that IR fluorescence is measured concentration of protein in solution, it is characterized in that: described glutaraldehyde solution adopts water or physiological saline or phosphate buffer to prepare as solvent.
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| CN2010101714903A CN101846625B (en) | 2010-05-13 | 2010-05-13 | Method for measuring concentration of protein in solution by using glutaraldehyde to induce infrared fluorescence |
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| CN2010101714903A CN101846625B (en) | 2010-05-13 | 2010-05-13 | Method for measuring concentration of protein in solution by using glutaraldehyde to induce infrared fluorescence |
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| CN101846625B true CN101846625B (en) | 2012-08-01 |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1335504A (en) * | 2001-03-28 | 2002-02-13 | 上海晶泰生物技术有限公司 | Protein chip for immunological analysis |
| CN1715924A (en) * | 2005-07-25 | 2006-01-04 | 福建农林大学 | Method for detecting tobacco mosaic virus using phycoerythrin fluorescent probe |
| WO2007148013A1 (en) * | 2006-06-19 | 2007-12-27 | Universite Pierre Et Marie Curie | Method for detecting and counting cells undergoing cytodieresis |
| CN101382482A (en) * | 2008-10-24 | 2009-03-11 | 西北工业大学 | Cell counting method |
-
2010
- 2010-05-13 CN CN2010101714903A patent/CN101846625B/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1335504A (en) * | 2001-03-28 | 2002-02-13 | 上海晶泰生物技术有限公司 | Protein chip for immunological analysis |
| CN1715924A (en) * | 2005-07-25 | 2006-01-04 | 福建农林大学 | Method for detecting tobacco mosaic virus using phycoerythrin fluorescent probe |
| WO2007148013A1 (en) * | 2006-06-19 | 2007-12-27 | Universite Pierre Et Marie Curie | Method for detecting and counting cells undergoing cytodieresis |
| CN101382482A (en) * | 2008-10-24 | 2009-03-11 | 西北工业大学 | Cell counting method |
Non-Patent Citations (2)
| Title |
|---|
| Jingbao Li, et al..Application of glutaraldehyde to in-cell Western assay of normalization.《Analytical Biochemistry》.2010,第398卷(第2期),254-256. * |
| 王文等.拓宽激光扫描共聚焦显微技术运用的新探索.《电子显微学报》.2002,第21卷(第1期),30-33. * |
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