CN102967496A - Preparation method of lymphocyte protein sample suitable for dimensional electrophoresis - Google Patents
Preparation method of lymphocyte protein sample suitable for dimensional electrophoresis Download PDFInfo
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Abstract
The invention discloses a preparation method of a lymphocyte protein sample suitable for dimensional electrophoresis, belonging to the technical field of disease pathogenesis researches. The preparation method comprises the following steps of: grinding a fresh rat spleen, filtering the ground fresh rat spleen in a 200-mesh manner, adding 4 DEG C 2ml of sodium chloride, transferring into a test tube, adding 2ml of a lymphocyte separation medium, and centrifuging to obtain a mononuclear cell; adding 2ml of double distilled water to rapidly crack RBCs (Red Blood Cells), adding 2ml of 1.8% sodium chloride to recover an isoosmotic state, washing the cells with 4 DEG C normal saline, and removing supernate; adding 2ml of 4 DEG C 5% cane sugar, mixing uniformly, and centrifuging to remove supernate; and drying a cell mass at room temperature, adding a hydration/lysis buffer to promote the dissolution of protein, ultrasonically breaking cells, and taking supernate, thus obtaining the lymphocyte protein sample suitable for the dimensional electrophoresis. With adoption of the preparation method disclosed by the invention for preparing the lymphocyte protein sample, the advantages of rapidness, simpleness and low cost are achieved, a high-quality dimensional electrophoretogram with good repeatability can be obtained, in addition, the subsequent biomass spectrometry protein analysis and identification researches are facilitated, and the preparation method can be widely applied to lymphocyte proteomics researches.
Description
Technical field
The invention belongs to disease incidence Mechanism Study technical field, be specifically related to a kind of lymphocyte protein sample preparation methods that is applicable to dielectrophoresis.
Background technology
Lymphocyte is one of immunocyte of human body, the important cells composition of immune response function.Account for leukocytic 20%-30% at human body.According to differences such as lymphocytic growth position, surface antigen, acceptor and functions, it is multiple lymphocyte can be divided into T lymphocyte and bone-marrow-derived lymphocyte and NK cell etc.Mature lymphocyte is brought into play important immunologic function in human body, a lot of diseases are all unusually relevant with number and function of T and B cells.The diseases such as picture infection, blood disease, tumour, anaphylactia, autoimmune disease, immunodeficiency are all closely related with lymphocyte.Along with scientific progress, the pathogenic process of lymphocyte and disease more and more interrelates.The enforcement of the Human Genome Project and propelling have made life science enter the genome times afterwards comprehensively, and one of sign of this transformation is exactly the rise of proteomics.Two-Dimensional Gel Electrophoresis (2-DE) is one of important technology of proteomics research, although the technical progress of proteomics research is at full speed, 2-DE remains one of method of carrying out the protein example separation.The preparation of protein sample is the key of 2-DE, also is the prerequisite of carrying out proteome analysis.Because contain salt and other impurity in lymphocytic separation and the cultivation, so that dielectrophoresis result's resolution is low, poor repeatability is difficult to obtain gratifying 2-DE collection of illustrative plates.
The existing commercial kit of lymphocyte protein preparation, but since expensive, be not suitable for sample study in enormous quantities.Brought difficulty for the further analysis of lymphocyte protein matter.Therefore it is very necessary setting up the lymphocyte protein extracting method that is applicable to electrophoretic analysis.
Summary of the invention
Order of the present invention provides a kind of lymphocyte protein sample preparation methods that has better quality and be applicable to dielectrophoresis.
The present invention includes the following step:
1) puts to death rat, separating spleen, get one of fresh spleen, filter with 200 mesh filter screens after grinding, 4 ℃ of 9% sodium chloride (physiological saline) 2ml dilution, obtain the lymphocyte suspension, and the immigration test tube, test tube is tilted 45 ° 1cm place on lymphocyte suspension liquid level, slowly add lymphocyte separation medium 2ml along the test tube tube wall, require lymphocyte separation medium not mix with the lymphocyte suspension, 2000rpm/min gradient centrifugation 20 minutes is drawn buffy coat, 2000rpm/min is centrifugal 10 minutes again, the Rats Spleen mononuclearcell that is precipitated;
2) distilled water 2ml is added in the Rats Spleen mononuclearcell of precipitation, jog 50 seconds after the RBC cracking, adds 1.8% sodium chloride 2ml (recovering to wait the state that oozes), centrifugal 10 minutes of 2000rpm/min immediately;
3) with step 2) repeat 1-2 time after, physiological saline is washed 2-3 time, removes supernatant, obtains that lymphocyte accounts for 97%, neutrophil leucocyte accounts for 3% cell mass;
4) in cell mass, add 4 ℃ 5% sucrose 2ml, mixing, 2000rpm/min horizontal centrifugal 10 minutes is removed supernatant;
5) with step 4) repeat 2-3 time (to remove the salinities such as sodium chloride of aqueous phase), the microscopically counting is with 5 * 10
7Individual lymphocyte is as an electrophoresis amount;
6) with step 5) lymphocyte of an electrophoresis amount moves on in the mortar, drying at room temperature 20 minutes, add aquation/lysis buffer 100 μ l (to promote the dissolving of albumen), the broken lymphocyte of Ultrasound Instrument, 1 second ultrasonic time, interval 1 second, power 8-9W is limpid to solution, centrifugal 30 minutes of 4 ° of C 15000r/min leave and take supernatant, namely obtain being applicable to the lymphocyte protein sample of dielectrophoresis.
Step 6) described aquation/lysis buffer is the solution that contains the saturated phenol 40mM of Tris, urea 7M, thiocarbamide 2M, CHAPS2%, DTT40mM, PMSF storage liquid 100 μ M, bromophenol blue 0.002% and IPG2% damping fluid.
The present invention adopts sucrose method washing lymphocyte, to remove the salinity such as aqueous phase sodium chloride, can avoid salts substances on the impact of dielectrophoresis.In aquation/lysis buffer of the present invention: urea can make the protein unfolding expose hydrophobic core, and is used the dissolubility that thiocarbamide can increase protein; Dithiothreitol (DTT) DTT Reduction of Disulfide, thus make protein reach consoluet state; The IPG damping fluid can be removed cyanate ion, accelerate the nucleic acid precipitation when centrifugal; PMSF can prevent that albumen is degraded.
It is quick, simple to adopt the present invention to prepare the lymphocyte protein sample, cost is low, can obtain the Two-dimensional Gel Electrophoresis of high-quality, good reproducibility, be convenient to the follow-up research of carrying out biological mass spectrometry Analysis and Identification albumen, the present invention can be widely used in the research of lymphocyte protein matter group, has higher practical value.
Description of drawings
Fig. 1 is Rats Spleen lymphocyte protein sample Two-dimensional Gel Electrophoresis
Fig. 2 is Human Lymphocytes protein sample Two-dimensional Gel Electrophoresis
Wherein: MW is protein molecular weight standard, and unit is kDa.
Embodiment
Experimental technique among the following embodiment if no special instructions, is conventional method.
Concentration among the following embodiment if no special instructions, is percent by volume (V/V).
Embodiment 1, preparation Rats Spleen lymphocyte protein dielectrophoresis sample also carry out Two-dimensional Electrophoresis Analysis
1) collects the experimental rat model of asthma spleen lymphocyte.Get one of spleen after putting to death rat, after fresh spleen grinds, filter with 200 mesh filter screens, with 4 ℃ of normal saline dilutions of 2ml, obtain the lymphocyte suspension, and immigration test tube, test tube is tilted 45 °, and 1cm place on lymphocyte suspension liquid level slowly adds lymphocyte separation medium (relative density 1.080) 2ml (requiring lymphocyte separation medium not mix with the lymphocyte suspension) along the test tube tube wall, 2000rpm/min gradient centrifugation 20 minutes is drawn buffy coat; 2000rpm/min is centrifugal 10 minutes again, the Rats Spleen mononuclearcell that is precipitated;
2) distilled water 2ml is added in the Rats Spleen mononuclearcell of precipitation, jog 50 seconds after the RBC cracking, adds 1.8% sodium chloride 2ml immediately, centrifugal 10 minutes of 2000rpm/min;
3) with step 2) repeat 1-2 time after, physiological saline is washed 2-3 time, removes supernatant, obtains that lymphocyte accounts for 97%, neutrophil leucocyte accounts for 3% cell mass;
4) in cell mass, add 4 ℃ 5% sucrose 2ml, mixing, 2000rpm/min horizontal centrifugal 10 minutes is removed supernatant;
5) with step 4) repeat 2-3 time, the microscopically counting is with 5 * 10
7Individual lymphocyte is as an electrophoresis amount;
6) with step 5) lymphocyte of an electrophoresis amount moves on in the mortar, drying at room temperature 20 minutes, add aquation/lysis buffer 100 μ l, promote the dissolving of albumen, the broken lymphocyte of Ultrasound Instrument, 1 second ultrasonic time, interval 1 second, power 8-9W is limpid to solution, centrifugal 30 minutes of 4 ° of C15000r/min, leave and take supernatant, namely obtain being applicable to the lymphocyte protein sample of dielectrophoresis;
7) with step 6) after the Rats Spleen lymphocyte protein that obtains was dissolved in aquation/lysis buffer, the centrifugal supernatant of leaving and taking obtained Rats Spleen lymphocyte protein dielectrophoresis sample liquid; Described aquation/lysis buffer aquation/lysis buffer is the solution that contains the saturated phenol 40mM of Tris, urea 7M, thiocarbamide 2M, CHAPS 2%, DTT40mM, PMSF storage liquid 100 μ M, bromophenol blue 0.002% and IPG2% damping fluid;
8) albumen that extracts by said process is measured protein concentration with Bradford method ultraviolet spectrophotometer, and the result shows the lymphocyte protein sample for dielectrophoresis obtained above, and protein concentration is 3-4ug/uL;
9) carry out as follows dielectrophoresis: (General Electric's Medical Group USA) carries out first to isoelectric focusing electrophoresis (IEF) at 20 ℃ to utilize Ettan IPGphor I I Isoelectric Focusing System.Deposition condition is: carry out isoelectric focusing electrophoresis at 20 ℃ with setting program, set 8000 volts of maximum voltages, electric current 50 μ A/gel, time 10.5h.The actual total voltage time is long-pending to be 42,000Vh.Then the Stationary pH gradient adhesive tape (IPG adhesive tape) after will focusing on is at 10mL level pad 1 (50mmol/L Tris-Cl damping fluid, pH8.8; 6mol/L urea; 30% glycerine, 0.02g/mL SDS, 0.01g/mL dithiothreitol (DTT)) middle balance 15min, then at 10ml level pad 2 (50mmol/L Tris-Cl damping fluid, pH8.8; 6mol/Lurea; 30% glycerine, 0.02g/mL SDS, 0.25g/mL iodoacetamide) middle balance 15min.Adhesive tape after the balance is transferred to second on gel, second utilizes Ettan DALTsix system to electrophoresis, and (General Electric's Medical Group USA) carries out at the 12.5%SDS-polyacrylamide gel, and electrophoresis is carried out in constant current, electric current 10mA/ glue, 300 volts of voltages, time 0.5h, electric current 20mA/ glue, 300 volts of voltages, time 12h when treating that bromophenol blue arrives the gel forward position to about 0.5cm, stops electrophoresis.Gel adopts silver nitrate method staining, the result as shown in Figure 1, the result shows that the lymphocyte protein sample separation is respond well, and obvious protein degradation does not occur.
Embodiment 2, preparation Human Lymphocytes protein sample also carry out Two-dimensional Electrophoresis Analysis
1) takes human blood 2ml, the EDTA anti-freezing;
2) use the 2ml normal saline dilution, through 4ml lymphocyte separation medium (relative density 1.077) gradient centrifugation, separate Human Lymphocytes;
3) through hypotonic splitting erythrocyte, cyclic washing is removed blood platelet, draws the mononuclearcell layer, 4 ℃, 2000rpm/min, centrifugal 5 minutes, wash 3 times with 4 ℃ of physiological saline after, remove supernatant;
4) add 4 ℃ 5% sucrose 2ml, mixing concussion 30 seconds, 2000rpm/min horizontal centrifugal 10 minutes is removed supernatant, 2-3 time repeatedly; The microscopically counting, 5 * 10
7Individual lymphocyte is that an electrophoresis amount is carried out packing;
5) cell mass was transferred in the mortar drying at room temperature 20 minutes, added the dissolving that aquation/lysate 100 μ l promote albumen;
6) Ultrasound Instrument smudge cells.1 second interval of ultrasonic time 1 second, power 8～9W is limpid to solution, and 4 ° of C centrifugal (15000r/min, 30 minutes) leave and take supernatant, namely obtain being applicable to the lymphocyte protein sample of dielectrophoresis;
7) with step 6) the Human Lymphocytes protein dissolution that obtains behind aquation/lysis buffer, centrifugal, leave and take supernatant, obtain Human Lymphocytes protein dielectrophoresis sample liquid; Described lysis buffer is for containing the saturated phenol 40mM of Tris, Urea 7M, and Thiourea 2M, CHAPS 2%, DTT 40mM, PMSF storage liquid 100 μ M, the solution of 0.002% bromophenol blue and 2%IPG damping fluid;
8) albumen that extracts by said process is measured protein concentration with Bradford method ultraviolet spectrophotometer, and the result shows the lymphocyte protein sample for dielectrophoresis obtained above, and protein concentration is 3-4ug/uL;
9) carry out as follows dielectrophoresis: (General Electric's Medical Group USA) carries out first to isoelectric focusing electrophoresis (IEF) at 20 ℃ to utilize Ettan IPGphor I I Isoelectric Focusing System.Deposition condition is: carry out isoelectric focusing electrophoresis at 20 ℃ with setting program, set 5000 volts of maximum voltages, electric current 50 μ A/gel, time 10.5h.The actual total voltage time is long-pending to be 42,000Vh.Then the Stationary pH gradient adhesive tape (IPG adhesive tape) after will focusing on is at 10mL level pad 1 (50mmol/L Tris-Cl damping fluid, pH8.8; 6mol/L urea; 30% glycerine, 0.02g/mL SDS, 0.01g/mL dithiothreitol (DTT)) middle balance 15min, then at 10ml level pad 2 (50mmol/L Tris-Cl damping fluid, pH8.8; 6mol/Lurea; 30% glycerine, 0.02g/mL SDS, 0.25g/mL iodoacetamide) middle balance 15min.Adhesive tape after the balance is transferred to second on gel, second utilizes Ettan DALTsix system to electrophoresis, and (General Electric's Medical Group USA) carries out at the 12.5%SDS-polyacrylamide gel, and electrophoresis is carried out in constant current, electric current 10mA/ glue, 300 volts of voltages, time 0.5h, electric current 20mA/ glue, 300 volts of voltages, time 12h when treating that bromophenol blue arrives the gel forward position to about 0.5cm, stops electrophoresis.Gel adopts silver nitrate method staining, the result as shown in Figure 2, the result shows that the lymphocyte protein sample separation is respond well, and obvious protein degradation does not occur.
Claims (2)
1. a lymphocyte protein sample preparation methods that is applicable to dielectrophoresis is characterized in that comprising the following steps:
1) puts to death rat, separating spleen, get one of fresh spleen, filter with 200 mesh filter screens after grinding, 4 ℃ of 9% sodium chloride 2ml dilution, obtain the lymphocyte suspension, and the immigration test tube, test tube is tilted 45 ° 1cm place on lymphocyte suspension liquid level, slowly add lymphocyte separation medium 2ml along the test tube tube wall, require lymphocyte separation medium not mix with the lymphocyte suspension, 2000rpm/min gradient centrifugation 20 minutes is drawn buffy coat, 2000rpm/min is centrifugal 10 minutes again, the Rats Spleen mononuclearcell that is precipitated;
2) distilled water 2ml is added in the Rats Spleen mononuclearcell of precipitation, jog 50 seconds after the RBC cracking, adds 1.8% sodium chloride 2ml immediately, centrifugal 10 minutes of 2000rpm/min;
3) with step 2) repeat 1-2 time after, physiological saline is washed 2-3 time, removes supernatant, obtains that lymphocyte accounts for 97%, neutrophil leucocyte accounts for 3% cell mass;
4) in cell mass, add 4 ℃ 5% sucrose 2ml, mixing, 2000rpm/min horizontal centrifugal 10 minutes is removed supernatant;
5) with step 4) repeat 2-3 time, the microscopically counting is with 5 * 10
7Individual lymphocyte is as an electrophoresis amount;
6) with step 5) lymphocyte of an electrophoresis amount moves on in the mortar, drying at room temperature 20 minutes, add aquation/lysis buffer 100 μ l, the broken lymphocyte of Ultrasound Instrument, 1 second ultrasonic time, interval 1 second, power 8-9W is limpid to solution, centrifugal 30 minutes of 4 ° of C 15000r/min leave and take supernatant, namely obtain being applicable to the lymphocyte protein sample of dielectrophoresis.
2. by the lymphocyte protein sample preparation methods that is applicable to dielectrophoresis claimed in claim 1, it is characterized in that step 6) described aquation/lysis buffer is the solution that contains the saturated phenol 40mM of Tris, urea 7M, thiocarbamide 2M, CHAPS 2%, DTT40mM, PMSF storage liquid 100 μ M, bromophenol blue 0.002% and IPG 2% damping fluid.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104075924A (en) * | 2014-07-15 | 2014-10-01 | 中国农业科学院农产品加工研究所 | Preparation and dielectrophoresis technology suitable for total protein of skeletal muscle of ruminants |
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| CN102645357A (en) * | 2012-03-27 | 2012-08-22 | 浙江大学 | Method for preparing spirulina dielectrophoresis analysis protein |
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Patent Citations (4)
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| SU1569650A1 (en) * | 1987-06-01 | 1990-06-07 | Киевский Медицинский Институт Им.Акад.А.А.Богомольца | Method of disclosing t- and b-lymphocytes in histological preparation |
| JP2007112732A (en) * | 2005-10-19 | 2007-05-10 | Tokyo Rika Kikai Kk | Method for solubilizing protein |
| CN102645357A (en) * | 2012-03-27 | 2012-08-22 | 浙江大学 | Method for preparing spirulina dielectrophoresis analysis protein |
| CN102721592A (en) * | 2012-07-12 | 2012-10-10 | 吉林大学 | Method for preparing aphid protein sample applicable to dielectrophoresis |
Non-Patent Citations (2)
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| 李善玉: "支气管哮喘大鼠淋巴细胞的差异表达蛋白组学研究", 《中国优秀博硕士学位论文全文数据库（博士）医药卫生科技辑》 * |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104075924A (en) * | 2014-07-15 | 2014-10-01 | 中国农业科学院农产品加工研究所 | Preparation and dielectrophoresis technology suitable for total protein of skeletal muscle of ruminants |
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Application publication date: 20130313 |