CN112782398A - Trace protein immunoblotting detection method - Google Patents
Trace protein immunoblotting detection method Download PDFInfo
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- CN112782398A CN112782398A CN202011581697.8A CN202011581697A CN112782398A CN 112782398 A CN112782398 A CN 112782398A CN 202011581697 A CN202011581697 A CN 202011581697A CN 112782398 A CN112782398 A CN 112782398A
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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
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/416—Systems
- G01N27/447—Systems using electrophoresis
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention relates to a trace protein immunoblotting detection method, which belongs to the field of protein detection, and is improved on the basis of the traditional protein immunoblotting detection method, the cell is directly lysed by using a sample buffer solution, the loss of protein in the traditional cell lysis process by using a lysate is avoided, the aperture of a glue making comb is adjusted to be 1.0-1.5mm, a detection signal is enhanced, and the defect that the total amount of protein donor cells is more than 10 in the prior art of protein immunoblotting technology is overcome6The defect of the single-component monoclonal antibody can be used for detecting the expression of a small amount of cell protein, and the detection range of the immunoblotting is effectively improved.
Description
Technical Field
The invention belongs to the field of protein detection, and particularly relates to a trace protein immunoblotting detection method.
Background
Western Blot, is a commonly used experimental method in molecular biology, biochemistry and immunogenetics. The basic principle is to stain a gel electrophoresis treated cell or biological tissue sample with a specific antibody. Information on the expression of a specific protein in the analyzed cell or tissue is obtained by analyzing the location and depth of staining. The western blotting method uses polyacrylamide gel electrophoresis, the detected substance is protein, the probe is antibody, and the secondary antibody is labeled for developing color. A protein sample separated by PAGE (polyacrylamide gel electrophoresis) is transferred to a solid support (e.g., nitrocellulose membrane) which adsorbs the protein in a non-covalent bond, andthe type of the polypeptide separated by electrophoresis and the biological activity thereof can be kept unchanged. Taking protein or polypeptide on a solid phase carrier as an antigen, carrying out immunoreaction with a corresponding antibody, then carrying out reaction with a second antibody labeled by enzyme or isotope, and carrying out substrate chromogenic or autoradiography to detect the protein component expressed by the specific target gene separated by electrophoresis. The existing Western blotting technology has high requirements on the cell quantity of total protein, generally more than 106One cell can extract enough protein for subsequent detection. In the actual experimental process, many experiments just collect enough cell quantity, so that the Western blotting experiment cannot be carried out, and the development of scientific research is hindered.
Disclosure of Invention
In view of the above, the present invention aims to provide a trace western blotting detection method.
In order to achieve the purpose, the invention provides the following technical scheme:
1. a trace protein immunoblotting detection method comprises the following steps,
(1) collecting cells to be detected, adjusting the number of the cells to be detected to be 500-;
(2) adding an equal-volume sample loading buffer solution into the cell lysate, uniformly mixing, and boiling to obtain a sample loading protein solution;
(3) preparing electrophoresis gel, pouring the gel into a gel making plate, inserting into a hole making comb, taking out the hole making comb after the gel is solidified, and forming a sample loading hole, wherein the width of the sample loading hole is 1.0-1.5 mm;
(4) adding the loading protein solution in the step (2) into the loading hole, and performing electrophoresis;
(5) then carrying out rotary die and antibody incubation, and finally detecting by using an ECL reagent.
As one preferable technical scheme, the lysis solution in the step (2) is a mixture of protease and phosphatase inhibitor.
As one of the preferable technical solutions, the gel in the step (3) is SDS-PAGE electrophoresis gel.
As one of the preferred technical schemes, the SDS-PAGE electrophoresis gel comprises 5% of concentrated gel and 10% of separation gel.
As one of the preferable technical proposal, the electrophoresis condition in the step (3) is that electrophoresis is performed for 15min at 100V, and then electrophoresis is performed to the bottom at 150V until the indicator is electrophoresed.
The invention has the beneficial effects that:
the sample buffer solution directly cracks cells, so that the loss of protein in the traditional cell cracking process of the lysate is avoided, and errors caused by inaccurate cell counting are overcome; by limiting the width of the sample loading hole to be 1.0-1.5mm, the detection signal is enhanced, and the defect that the total amount of protein donor cells is more than 10 in the prior Western blotting technology is overcome6Due to the defects, a small number of cells (thousands to tens of thousands) can also detect the expression condition of the target protein by using the Western blotting technology, thereby greatly improving the application range of the Western blotting technology and being beneficial to the development of basic research.
Drawings
FIG. 1 shows actin measured in different numbers of hematopoietic stem cells and hematopoietic progenitor cell populations, 1 and 2 for hematopoietic progenitor cells measured in 3mm loading wells, 3 for standard protein, 4-6 for hematopoietic progenitor cells measured in 1.5mm loading wells, and 7-9 for hematopoietic stem cells measured in 1.5mm loading wells;
FIG. 2 shows the detection of EIF4G, actin and phosphorylated protein pS6 in different numbers of hematopoietic stem cells and hematopoietic progenitor cell populations.
Detailed Description
Hereinafter, specific embodiments of the present invention will be described in detail. Through the embodiments, the present invention can be more clearly understood by scientific researchers, and certain changes and modifications can be made on the basis of the embodiments to obtain different research effects of the experimental methods in the following embodiments, which are conventional methods unless otherwise specified. The reagents involved in the experimental process are all conventional reagents, and the use of the reagents is all referred to the product use instruction.
Example 1
Sorting Hemopoietic Stem Cells (HSC) and progenitor cells (HP) by a flow cytometer, transferring the HSC and the HP obtained by sorting into EP tubes respectively, wherein the number of the cells in each EP tube is 20000, and centrifuging for 5min at 4 ℃ and 500 g. Part of the liquid was removed so that the ratio of the number of cells to the remaining liquid was 500: 1ul, record residual liquid volume. Agitation of the bottom cell pellet is avoided during removal of the liquid. Adding a mixed solution of 2 Xloading buffer, 1000 Xprotease and 1000 Xphosphatase inhibitor in a volume ratio of 1000:1:1 to lyse cells to obtain cell lysate. Adding equal volume of 2 Xloading buffer solution into cell lysate, mixing uniformly, and boiling for 5min to obtain loading protein solution. Preparing SDS-PAGE electrophoresis gel comprising 5% concentrated gel and 10% separation gel, pouring the gel into a gel making plate, inserting a hole making comb with the tooth width of 1.5mm, taking a conventional 3mm gel making comb as a control, taking out the hole making comb after the gel is solidified, and preparing the sample loading gel plate.
According to the relation between the number of cells and the volume of the loading protein solution, 1000 and 500 hematopoietic progenitor cells are respectively added into loading holes 1 and 2 with the length of 3mm, standard protein is added into loading hole 3 with the length of 3mm, 2000, 1000 and 500 hematopoietic progenitor cells are respectively added into loading holes 4-6 with the length of 1.5mm, and 2000, 1000 and 500 hematopoietic stem cells are respectively added into loading holes 7-9 with the length of 1.5 mm. And (3) performing 100V electrophoresis, and after the protein enters the separation gel, boosting the pressure to 150V until the indicator reaches the bottom of the gel. And transferring the model according to the standard flow, and then performing actin antibody incubation in a ratio of 1:1000 to enhance the ECL reagent for detection. The results are shown in figure 1, the loading hole obtained by the glue making comb with the thickness of 1.5mm has obvious protein bands after electrophoresis, the number of cells which can be detected can be obviously reduced, and the protein in the hematopoietic stem cells is obviously less than that in the hematopoietic progenitor cells, which is consistent with the report of related literatures, and the reliability of the results is also confirmed.
Loading wells of 1.5mm each were prepared according to the above method, and 500, 1000 and 2000 hematopoietic stem cells, 500, 1000, 2000, 3000 steady-state hematopoietic progenitor cells and hematopoietic progenitor cells after hematopoietic Stem Cell Factor (SCF) stimulation were loaded, respectively. And (3) performing 100V electrophoresis, and after the protein enters the separation gel, boosting the pressure to 150V until the indicator reaches the bottom of the gel. The assay was performed using ECL-enhanced reagents by performing a 1:1000 ratio transfer and incubation with EIF4G, actin and p-S6 antibodies, respectively, according to standard protocols. As shown in FIG. 2, EIF4G, actin and p-S6 in 500, 1000 and 2000 cells were detected in 1.5mm of air, and the reliability and repeatability of the method were confirmed.
Finally, the above embodiments are only intended to illustrate the technical solutions of the present invention and not to limit the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it will be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions, and all of them should be covered by the claims of the present invention.
Claims (5)
1. A trace protein immunoblotting detection method is characterized in that,
(1) collecting cells to be detected, adjusting the number of the cells to be detected to be 500-;
(2) adding an equal volume of 2 multiplied sample buffer solution into cell lysate, uniformly mixing and boiling to obtain a sample protein solution;
(3) preparing electrophoresis gel, pouring the gel into a gel making plate, inserting into a hole making comb, taking out the hole making comb after the gel is solidified, and forming a sample loading hole, wherein the width of the sample loading hole is 1.0-1.5 mm;
(4) adding the loading protein solution in the step (2) into the loading hole, and performing electrophoresis;
(5) then carrying out rotary die and antibody incubation, and finally detecting by using an ECL reagent.
2. The assay of claim 1, wherein the loading buffer in step (2) is 2 x.
3. The detection method according to claim 1, wherein the electrophoresis gel in the step (3) is SDS-PAGE electrophoresis gel.
4. The assay of claim 3 wherein said SDS-PAGE electrophoresis gel comprises 5% concentrated gel and 10% separation gel.
5. The detection method according to claim 1, wherein the electrophoresis in step (4) is performed under the conditions of electrophoresis at 100V for 15min and then at 150V until the indicator is electrophoresed to the bottom.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310432602.3A CN116466071A (en) | 2020-12-28 | 2020-12-28 | Application of loading buffer solution and trace western blotting detection method |
| CN202011581697.8A CN112782398A (en) | 2020-12-28 | 2020-12-28 | Trace protein immunoblotting detection method |
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| CN202011581697.8A CN112782398A (en) | 2020-12-28 | 2020-12-28 | Trace protein immunoblotting detection method |
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1189839A (en) * | 1995-05-01 | 1998-08-05 | 美国汤姆森有限公司 | Compsns. and methods for detecting and treating acquired immunodeficiency syndrome |
| CN102087292A (en) * | 2009-12-02 | 2011-06-08 | 国家纳米科学中心 | Microfluidic immune imprinting chip and preparation method and application thereof |
| CN103060421A (en) * | 2013-01-22 | 2013-04-24 | 上海市内分泌代谢病研究所 | Autophagy monitoring method for fat cells |
| CN103987842A (en) * | 2011-09-30 | 2014-08-13 | 葛兰素史密斯克莱有限责任公司 | Methods of treating cancer |
| CN205749136U (en) * | 2016-05-15 | 2016-11-30 | 天津纽威特橡胶制品股份有限公司 | A kind of stadiums plastic cement race track detecting device for pressure strength |
| CN106480182A (en) * | 2016-10-12 | 2017-03-08 | 重庆医科大学附属第医院 | A kind of method of testing to spinal metastasis impact cell for polymethyl methacrylate |
| CN111826419A (en) * | 2020-07-06 | 2020-10-27 | 重庆生命知源科技有限公司 | Library-establishing sequencing method suitable for RIP (RIP-induced plasticity) experiment of trace cells |
-
2020
- 2020-12-28 CN CN202310432602.3A patent/CN116466071A/en not_active Withdrawn
- 2020-12-28 CN CN202011581697.8A patent/CN112782398A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1189839A (en) * | 1995-05-01 | 1998-08-05 | 美国汤姆森有限公司 | Compsns. and methods for detecting and treating acquired immunodeficiency syndrome |
| CN102087292A (en) * | 2009-12-02 | 2011-06-08 | 国家纳米科学中心 | Microfluidic immune imprinting chip and preparation method and application thereof |
| CN103987842A (en) * | 2011-09-30 | 2014-08-13 | 葛兰素史密斯克莱有限责任公司 | Methods of treating cancer |
| CN103060421A (en) * | 2013-01-22 | 2013-04-24 | 上海市内分泌代谢病研究所 | Autophagy monitoring method for fat cells |
| CN205749136U (en) * | 2016-05-15 | 2016-11-30 | 天津纽威特橡胶制品股份有限公司 | A kind of stadiums plastic cement race track detecting device for pressure strength |
| CN106480182A (en) * | 2016-10-12 | 2017-03-08 | 重庆医科大学附属第医院 | A kind of method of testing to spinal metastasis impact cell for polymethyl methacrylate |
| CN111826419A (en) * | 2020-07-06 | 2020-10-27 | 重庆生命知源科技有限公司 | Library-establishing sequencing method suitable for RIP (RIP-induced plasticity) experiment of trace cells |
Non-Patent Citations (2)
| Title |
|---|
| XIONGWEI CAI 等: "A Western Blotting Protocol for Small Numbers of Hematopoietic Stem Cells", 《JOURNAL OF VISUALIZED EXPERIMENTS》 * |
| 张蕾 等: "《生物化学实验指导》", 31 August 2011 * |
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