CN101004384A - Raman spectrum method for detecting surface reinforcement of protein group - Google Patents
Raman spectrum method for detecting surface reinforcement of protein group Download PDFInfo
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Abstract
本发明属于蛋白质分析检测领域，特别涉及一种利用表面增强拉曼光谱在印迹固定化基质上直接对蛋白质进行检测的方法。包括处理蛋白质样品、提取蛋白质组；进行蛋白质电泳，将蛋白质混合物分离；进行蛋白质印迹，将已分离的蛋白质转移到固定基质上；进行银溶胶染色，显示出分离的蛋白质条带或蛋白质点；进行拉曼光谱检测，得到待测蛋白质的拉曼指纹图谱等步骤。根据蛋白质的拉曼指纹图谱，可检测出蛋白质组中特定的蛋白质。本发明可用于蛋白质印迹后，固定基质上蛋白质的直接检测，也可用于免疫复合物的检测。本发明在蛋白质组研究的高通量检测方面具有巨大的应用潜力，可为蛋白质组研究提供新的检测手段。
The invention belongs to the field of protein analysis and detection, in particular to a method for directly detecting protein on imprinted immobilized substrates by using surface-enhanced Raman spectroscopy. Including processing protein samples and extracting proteomes; performing protein electrophoresis to separate protein mixtures; performing Western blotting to transfer the separated proteins to a fixed matrix; performing silver sol staining to display separated protein bands or protein spots; conducting Raman spectrum detection, obtaining the Raman fingerprint of the protein to be tested and other steps. Specific proteins in the proteome can be detected based on the Raman fingerprint of the protein. The invention can be used for the direct detection of the protein on the immobilized matrix after western blotting, and can also be used for the detection of the immune complex. The invention has great application potential in high-throughput detection of proteome research, and can provide a new detection means for proteome research.
Description
Technical field
The invention belongs to the protein analysis detection range, particularly a kind of method of utilizing Surface enhanced raman spectroscopy on the trace immobilization matrix, directly protein to be detected.
Background technology
Along with finishing of the Human Genome Project, the research of protein group becomes focus in recent years, though because DNA can provide a lot of information, protein is only whole embodiments of cell function.Protein group refers to all protein and the existing way thereof that full gene is expressed, and is a complete set of protein that a kind of cell, tissue or complete biosome are had on specific space-time.Proteomics is different from traditional single protein or the research of certain class protein, and what its was studied is whole protein and dynamic rule thereof in the system, comprises the research of the constitutive protein matter interphase interaction of protein group.Surface enhanced raman spectroscopy (SERS) technology has special advantages in the biological big molecular composition configuration aspects of research, its original position and real-time the detection and high characteristics such as detection sensitivity, giving the biological macromolecular structure of research and interacting provides tempting solution, and it also provides new power for the development of fields such as immunology, living things catalysis, drug design.The coupling of Surface enhanced raman spectroscopy technology and other analysis means not only will play a significant role in proteomics research, and can promote the development of other related disciplines.
The proteome research progress is very rapid, though basic theory or technical method, all constantly progressive and perfect.The new technology of Analysis and Identification protein, new method constantly occurs.At present, protein identification techniques mainly contains: image analysis technology, microsequencing technology reach the technology relevant with mass spectrum etc.
The technology close with the present invention is disclosed Chinese patent on September 24th, 2003 " surface strength laman scattering mark immunodetection ", publication number CN1444045A.It discloses the immunoassay technology with the reaction of Surface enhanced raman spectroscopy spectral detection antigen and antibody specific.Through formation assembly, prepare the SERS immunoglobulin g-gold, immune recombination reaction, processes such as silver dyeing are with Raman spectrum view certification mark immune complex.This patent has overcome the operation steps complexity of this surface strength laman scattering mark immunoassay technology and the shortcomings such as narrow range of detected object, can improve the efficient of protein detection to a great extent.
Summary of the invention
The purpose of this invention is to provide a kind of means of on the trace immobilization matrix, utilizing surface-enhanced Raman directly non-marked protein or labelled protein to be detected, improve the sensitivity of protein detection, have limitation such as operation steps complexity, detection efficiency are low to overcome the traditional detection method of protein.
The present invention makees silver-colored dye liquor at the bottom of utilizing the SERS active group, combines biological silver and dyes advantage with two aspects of SERS.Silver both dyed can keep good development contrast, can realize detection again to different proteins band or protein spots, easy and simple to handle, thereby can obtain the Raman finger-print of multiple proteins, also can identify the protein of differential expression apace, provide new detection means for high throughput protein detects.Simultaneously, Raman spectrum belongs to characteristic spectrum, and the molecule of different structure has different vibrational spectra peaks, so this method can improve the sensitivity of protein detection greatly.
The Raman spectrum method for detecting surface reinforcement of protein group of the present invention is as follows: (a) handle protein example, extract protein group; (b) carry out protein electrophorese, protein mixture is separated into band or protein spots; (c) carry out Western blotting, the protein band or the protein spots that have separated are transferred on the immobilization matrix; (d) carry out silver sol dyeing, demonstrate the protein band or the protein spots of separation; (e) carry out Raman spectrum and detect, obtain the Raman finger-print of testing protein.
Said protein electrophorese is the technology that protein mixture is separated by molecular weight or isoelectric point in the said method, can be electrophoretic techniques commonly used such as polyacrylamide gel electrophoresis or sds polyacrylamide gel electrophoresis or isoelectric focusing electrophoresis or dielectrophoresis etc., used medium be polyacrylamide gel, Ago-Gel or isoelectric focusing gel; Said Western blotting be all protein that will separate behind the electrophoresis by electrotransfer, promptly under effect of electric field, protein is convenient to detection on transferring to fixedly matrix from gel; Fixedly matrix can be trace immobilization matrix commonly used such as nitrocellulose filter, polyvinylene double oxide (PVDF) film, nylon membrane, DEAE-cellulose paper or cellulose phosphate paper; Said silver sol dyeing is that the fixedly matrix that obtains after protein is shifted is dipped in the silver sol, and silver sol is that liquor argenti nitratis ophthalmicus is made by the sodium citrate reduction under little state that boils; At last, can directly carry out qualitative detection by the protein band or the protein spots of Raman spectrometer after to trace, or with the antibody effect after silver dye, detect the antigenicity of specified protein in the protein group again with Raman spectrometer.
The testing process of the Raman spectrum method for detecting surface reinforcement of protein group of the present invention can be explained with Fig. 1: among Fig. 1,1 is electrophoretic medium; 2 is immobilization matrix; 3 is protein band; 4 is protein spots; 5 is the used LASER Light Source of Raman spectrometer, and 6 collect the Surface enhanced raman spectroscopy of protein for Raman spectrometer; 7 expression protein electrophorese processes; 8 expression Western blotting processes, 9 dyeing of expression silver sol and Raman detection processes.
Above-mentioned protein group of carrying out gel electrophoresis can be a bovine serum albumin matter group, ox muscle protein group, and the protein group that human serum protein group or other any suitable protein electrophorese separate can be carried out multiple-stage treatment to sample before the electrophoresis, removes impurity.
Above-mentioned gel electrophoresis can be rectilinear or horizontal, continuously or the discontinuous polyethylene acrylamide gel electrophoresis.After preparing glue, sample on the protein group sample in well, under the condition of energising, according to the difference of molecular weight and molecule carried charge, is obtained in the gel of protein group sample in being immersed in damping fluid separating; Above-mentioned gel electrophoresis also can be a sds page, and the protein group sample obtains separating according to the difference of molecular weight; Above-mentioned gel electrophoresis also can be an isoelectric focusing electrophoresis, can be with tubulose gel or vertical slab gel, and the protein group sample obtains separating in carrier ampholyte PH gradient or the solid phase PH gradient media difference because of isoelectric point; Above-mentioned gel electrophoresis can also be a dielectrophoresis, and first to being isoelectric focusing, and second to sds polyacrylamide gel electrophoresis, and protein example is pressed molecular weight earlier, obtains by the difference of isoelectric point separating again.
Above-mentioned Western blotting can be an electroblotting, and under effect of electric field, the protein after gel electrophoresis separates is transferred in the electrophoretic blotting groove fixedly on the matrix, and electroblotting can be vertical trench or half dry type electroblotting.
Above-mentioned silver sol dyeing is to be dipped in the silver sol shifting the blotting membrane (immobilization matrix) of going up protein, silver sol is that liquor argenti nitratis ophthalmicus is made by the sodium citrate reduction under little state that boils, Nano silver grain is spherical in shape mostly, mean grain size is 70 nanometers, and soak time is 10-60 minute.
Above-mentioned Raman detection is carried out after silver sol dyeing, can directly detect institute's protein of interest particle or protein band on the blotting membrane, used spectrometer can be the burnt Raman spectrometer of copolymerization, and the wavelength coverage that can adopt excitation source is 400～1000nm.
Above-mentioned Raman detection also can be immersed in the blotting membrane behind the electroblotting earlier in the solution that contains one or more antibody, incubation one hour, carry out silver sol dyeing again, afterwards protein spots to be measured or protein band are carried out Raman detection, detect the antigenicity of testing protein according to the surface increasing Raman spectrum figure of immune complex; Also can be with the first incubation one hour in containing the solution of one or more labelled antibodies of the blotting membrane behind the electroblotting, carry out silver sol dyeing again, afterwards protein spots to be measured or protein band are carried out Raman detection, detect the antigenicity of testing protein according to the surface increasing Raman spectrum figure of antibody labeling thing.
The protein that said method detected is that (antibody is behind the label mark after adding antibody or labelled antibody behind non-labelled protein (embodiment 1 and embodiment 2) or the trace, its Raman signal is stronger than the signal of antibody itself, be more conducive to detect, embodiment 3) protein complex.
According to the Raman finger-print of protein, can detect particular proteins or protein complex in the protein group.The present invention has huge application potential aspect the high throughput testing of proteome research, can be proteome research new detection means is provided.The present invention's protein group change of Expression under influence that testing environment is expressed protein group and disease situation has huge application potential, provides possibility for understanding protein structure and function relationship.
Description of drawings
Fig. 1: the Raman spectrum method for detecting surface reinforcement step synoptic diagram of the described protein group of this patent;
The surface-enhanced Raman figure of Fig. 2: the embodiment 1 described bovine serum albumin(BSA) that from bovine serum albumin matter group, obtains;
The surface-enhanced Raman figure of Fig. 3: the embodiment 2 described myoglobins that from ox muscle protein group, obtain;
The surface-enhanced Raman figure of Fig. 4: the embodiment 3 described protein interactions that from the human serum protein group, obtain.
Embodiment
Embodiment 1: the SERS of bovine serum albumin(BSA) detects in the bovine serum albumin matter group
1) carry out before the electrophoresis, carry out the multiple-stage treatment of sample earlier: comprise clasmatosis, prevent proteolysis, precipitating proteins, step such as clear the pollution off is separated all protein in the cell with this, obtains bovine serum albumin matter group;
2) preparation sample buffer: 1.6mL buffering storage liquid (1mol/LTris-HCl, PH6.8), 4mol 10%SDS, the 1mL beta-mercaptoethanol, 2.5mL 87% glycerine, the 0.1mg bromophenol blue is diluted to 20mL with distilled water, 4 ℃ of preservations;
3) sample is carried out the vertical discontinuous electro-phoresis of sds page, the gel final concentration is 7.5%, and applied sample amount is 10 μ L, treats that the bromophenol blue forward position stops electrophoresis when arriving the electrophoresis tank bottom;
4) carry out half dry type protein electrotransfer, blotting membrane is selected nitrocellulose filter for use, and used transfering buffering liquid is the Tris damping fluid, and PH is 10.4, and be 2 hours transfer time;
5) immediately the blotting membrane after the protein transfer is immersed in the middle cleaning of Tris-HCl damping fluid (PH7.10) 10 minutes, afterwards blotting membrane is immersed in the silver sol of new preparation, silver sol is made by the sodium citrate method, and dyeing time is 30 minutes;
6) after dyeing finishes, blotting membrane is immersed in the middle cleaning of Tris-HCl damping fluid (PH7.10) 3 times, each 10 minutes, cleans each 10 minutes 3 times with three distilled water afterwards;
7) compare with time high-molecular-weight protein standard, find out the bovine serum albumin(BSA) band, carry out Raman detection, the burnt Raman spectrometer of the Renishaw-1000 type copolymerization that can use Britain's Reinshaw (Renishaw) company to produce.It adopts excitation source is air cooling Argon ion laser (Spectra-Physics Model 163-C4260), excitation wavelength 514.5nm.The laser instrument peak power output is 20mW, is equipped with attenuator, and confocal microscope system can be the German Leica DMLM of company type microscope, uses 50 object lens, and after laser focused on through microscopic system, the spot diameter size was 1 μ m.Data analysis is a platform with GRAMS/32 (Galatics), handles with Renishawv1.3WIRE software.
8) obtain the SERS characteristic spectrum (on Fig. 2) of bovine serum albumin(BSA), wherein 1650cm
-1, 1289cm
-1Point out amido link I and amido link II respectively into bovine serum albumin(BSA), the two representative be the alpha helical conformation of this albumen.In addition, 1605cm
-1, 1337cm
-1, 1002cm
-1, 826cm
-1Point out respectively and be side chain amino acid tyrosine, the vibration of tryptophane and phenylalanine.Compare with the common Raman figure (curve below among Fig. 2) of bovine serum albumin(BSA), the amido link peak position does not almost change.Relatively as seen two curves (Fig. 2) in the spectrogram have very big difference shown in the intensity at the spectrum peak shown in the following surface curve and quantity and the last surface curve, and this is owing to due to the interaction that does not have bovine serum albumin(BSA) and Nano silver grain.By with the interaction of Nano silver grain, under the low concentration situation, just can obtain the bovine serum albumin(BSA) Raman signal, operating process is simple, and the repeatability of spectrogram better, proves the superiority of the present invention aspect the qualitative detection of simple protein.
Embodiment 2: the SERS of myoglobins detects in the ox muscle protein group
As each step operation of example 1, different is, and what to obtain in the step 1) is ox muscle protein group, and blotting membrane is selected polyvinylene double oxide (PVDF) film for use, and the silver sol dyeing time is 20 minutes.Obtain the SERS spectrum (Fig. 3) of myoglobins, amido link (1229cm
-1), side chain amino acid (1263,1002cm
-1) and protoheme (1585,1399,1374,1313cm
-1) vibration appear on the spectrogram 3.Myoglobins belongs to compound protein, protoheme links to each other with the protein portion of myoglobins by non-covalent bond as prothetic group, and protoheme and myoglobins protein with preponderate during the competition of Nano silver grain combines, so do not observe the vibration of protein acid amides I, the spectrum that obtains mainly is the vibrational spectrum of protoheme.This spectrogram has reflected the feature Raman vibration of myoglobins, proves that the present invention can the qualitative detection compound protein.
Embodiment 3: the SERS of IgG detects in the human serum protein group
As each step operation of example 1, different is, and what to obtain in the step 1) is the human serum protein group; In step 2), what carry out in the step 3) is isoelectric focusing electrophoresis, the used carrier ampholyte is Ampholine, the PH scope is 3.5～10, blotting membrane is selected nitrocellulose filter for use; After the trace washing, nitrocellulose filter is immersed in 2000 times of dilute solutions of biotin labeled anti-human IgG in the step 4), incubated at room one hour, carrying out the silver sol dyeing time again is 40 minutes.At isoelectric point place near human IgG, obtained the SERS spectrogram (Fig. 4) of biotin molecule vibration, this result proves that interaction has taken place for biotin labeled anti-human IgG and human IgG, the also existence of IgG in reference's haemocyanin group is so the present invention also can be used for the immunoassay behind the Western blotting.
Claims (7)
1, the Raman spectrum method for detecting surface reinforcement of protein group comprises the steps:
(a) handle protein example, extract protein group; (b) carry out protein electrophorese, protein mixture is separated into band (3) or protein spots (4); (c) carry out Western blotting, the protein band (3) that separated or protein spots (4) are transferred to fixedly on the matrix (2); (d) carry out silver sol dyeing, demonstrate the protein band (3) or the protein spots (4) of separation; (e) carry out Raman spectrum and detect, obtain the Raman finger-print of testing protein.
2, the Raman spectrum method for detecting surface reinforcement of protein group as claimed in claim 1, it is characterized in that: protein electrophorese is polyacrylamide gel electrophoresis, sds polyacrylamide gel electrophoresis, isoelectric focusing electrophoresis or dielectrophoresis, and used medium is polyacrylamide gel, Ago-Gel or isoelectric focusing gel.
3, the Raman spectrum method for detecting surface reinforcement of protein group as claimed in claim 1 is characterized in that: add antibody or labelled antibody after carrying out Western blotting, and then carry out elargol dyeing.
4, the Raman spectrum method for detecting surface reinforcement of protein group as claimed in claim 1 is characterized in that: fixedly matrix (2) is nitrocellulose filter, polyvinylene double oxide film, nylon membrane, DEAE-cellulose paper or cellulose phosphate paper.
5, the Raman spectrum method for detecting surface reinforcement of protein group as claimed in claim 1 is characterized in that: Western blotting adopts vertical trench or half dry type electroblotting.
6, the Raman spectrum method for detecting surface reinforcement of protein group as claimed in claim 1, it is characterized in that: silver sol dyeing is to be dipped in the silver sol shifting the blotting membrane of going up protein, silver sol is that liquor argenti nitratis ophthalmicus is made by the sodium citrate reduction under little state that boils, and soak time is 10～60 minutes.
7, the Raman spectrum method for detecting surface reinforcement of protein group as claimed in claim 1 is characterized in that: used Raman spectrometer is the burnt Raman spectrometer of copolymerization, and the wavelength coverage that detects the excitation source that adopts is 400～1000nm.
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