CN114544738A - Electrophoresis buffer solution - Google Patents
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- CN114544738A CN114544738A CN202210135847.5A CN202210135847A CN114544738A CN 114544738 A CN114544738 A CN 114544738A CN 202210135847 A CN202210135847 A CN 202210135847A CN 114544738 A CN114544738 A CN 114544738A
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- 238000001962 electrophoresis Methods 0.000 title claims abstract description 83
- 239000007853 buffer solution Substances 0.000 title claims abstract description 53
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims abstract description 44
- 239000007983 Tris buffer Substances 0.000 claims abstract description 24
- 239000012153 distilled water Substances 0.000 claims abstract description 24
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 24
- 239000011780 sodium chloride Substances 0.000 claims abstract description 22
- 239000003109 Disodium ethylene diamine tetraacetate Substances 0.000 claims abstract description 14
- ZGTMUACCHSMWAC-UHFFFAOYSA-L EDTA disodium salt (anhydrous) Chemical compound [Na+].[Na+].OC(=O)CN(CC([O-])=O)CCN(CC(O)=O)CC([O-])=O ZGTMUACCHSMWAC-UHFFFAOYSA-L 0.000 claims abstract description 14
- 235000019301 disodium ethylene diamine tetraacetate Nutrition 0.000 claims abstract description 14
- 239000002994 raw material Substances 0.000 claims abstract description 7
- 239000000243 solution Substances 0.000 claims description 28
- 239000000872 buffer Substances 0.000 claims description 26
- GRYLNZFGIOXLOG-UHFFFAOYSA-N Nitric acid Chemical compound O[N+]([O-])=O GRYLNZFGIOXLOG-UHFFFAOYSA-N 0.000 claims description 19
- 229910017604 nitric acid Inorganic materials 0.000 claims description 19
- 239000012146 running buffer Substances 0.000 claims description 5
- 150000001875 compounds Chemical class 0.000 claims 1
- 238000002474 experimental method Methods 0.000 abstract description 29
- 210000002966 serum Anatomy 0.000 abstract description 20
- 229920006221 acetate fiber Polymers 0.000 abstract description 8
- 229960000796 barbital sodium Drugs 0.000 abstract description 5
- 238000011160 research Methods 0.000 abstract 1
- 239000010408 film Substances 0.000 description 53
- 239000011521 glass Substances 0.000 description 15
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 14
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 12
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 12
- 230000000052 comparative effect Effects 0.000 description 11
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 10
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 10
- 229940125717 barbiturate Drugs 0.000 description 10
- 238000002791 soaking Methods 0.000 description 10
- 229910052708 sodium Inorganic materials 0.000 description 10
- 239000011734 sodium Substances 0.000 description 10
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 9
- 239000007788 liquid Substances 0.000 description 9
- 239000012528 membrane Substances 0.000 description 9
- 238000002360 preparation method Methods 0.000 description 9
- 239000003513 alkali Substances 0.000 description 7
- LXAHHHIGZXPRKQ-UHFFFAOYSA-N 5-fluoro-2-methylpyridine Chemical compound CC1=CC=C(F)C=N1 LXAHHHIGZXPRKQ-UHFFFAOYSA-N 0.000 description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 6
- 229960000583 acetic acid Drugs 0.000 description 6
- 239000003153 chemical reaction reagent Substances 0.000 description 6
- 238000004043 dyeing Methods 0.000 description 6
- 239000012362 glacial acetic acid Substances 0.000 description 6
- 229910052742 iron Inorganic materials 0.000 description 6
- 238000000034 method Methods 0.000 description 6
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 6
- 102000009027 Albumins Human genes 0.000 description 5
- 108010088751 Albumins Proteins 0.000 description 5
- 101100313477 Arabidopsis thaliana THE1 gene Proteins 0.000 description 5
- 102000006734 Beta-Globulins Human genes 0.000 description 5
- 108010087504 Beta-Globulins Proteins 0.000 description 5
- 108010017384 Blood Proteins Proteins 0.000 description 5
- 102000004506 Blood Proteins Human genes 0.000 description 5
- 101100412671 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) RGA1 gene Proteins 0.000 description 5
- 239000003814 drug Substances 0.000 description 5
- 108010074605 gamma-Globulins Proteins 0.000 description 5
- 230000002159 abnormal effect Effects 0.000 description 4
- 238000007598 dipping method Methods 0.000 description 4
- 238000005370 electroosmosis Methods 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 4
- 239000002245 particle Substances 0.000 description 4
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- FTOAOBMCPZCFFF-UHFFFAOYSA-N 5,5-diethylbarbituric acid Chemical compound CCC1(CC)C(=O)NC(=O)NC1=O FTOAOBMCPZCFFF-UHFFFAOYSA-N 0.000 description 3
- 101100495769 Caenorhabditis elegans che-1 gene Proteins 0.000 description 3
- 206010053567 Coagulopathies Diseases 0.000 description 3
- 241000283973 Oryctolagus cuniculus Species 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- 229920002301 cellulose acetate Polymers 0.000 description 3
- 230000035602 clotting Effects 0.000 description 3
- 229940079593 drug Drugs 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 239000012192 staining solution Substances 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 108010044091 Globulins Proteins 0.000 description 2
- 102000006395 Globulins Human genes 0.000 description 2
- 208000034578 Multiple myelomas Diseases 0.000 description 2
- 206010035226 Plasma cell myeloma Diseases 0.000 description 2
- 108010045362 Serum Globulins Proteins 0.000 description 2
- 102000005686 Serum Globulins Human genes 0.000 description 2
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- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
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- 238000013508 migration Methods 0.000 description 2
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- 238000000926 separation method Methods 0.000 description 2
- NOIIUHRQUVNIDD-UHFFFAOYSA-N 3-[[oxo(pyridin-4-yl)methyl]hydrazo]-N-(phenylmethyl)propanamide Chemical compound C=1C=CC=CC=1CNC(=O)CCNNC(=O)C1=CC=NC=C1 NOIIUHRQUVNIDD-UHFFFAOYSA-N 0.000 description 1
- 108060003951 Immunoglobulin Proteins 0.000 description 1
- 208000032382 Ischaemic stroke Diseases 0.000 description 1
- JLVVSXFLKOJNIY-UHFFFAOYSA-N Magnesium ion Chemical compound [Mg+2] JLVVSXFLKOJNIY-UHFFFAOYSA-N 0.000 description 1
- 101710085938 Matrix protein Proteins 0.000 description 1
- 101710127721 Membrane protein Proteins 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- VJMAITQRABEEKP-UHFFFAOYSA-N [6-(phenylmethoxymethyl)-1,4-dioxan-2-yl]methyl acetate Chemical compound O1C(COC(=O)C)COCC1COCC1=CC=CC=C1 VJMAITQRABEEKP-UHFFFAOYSA-N 0.000 description 1
- 239000012445 acidic reagent Substances 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 229960002319 barbital Drugs 0.000 description 1
- HNYOPLTXPVRDBG-UHFFFAOYSA-N barbituric acid Chemical compound O=C1CC(=O)NC(=O)N1 HNYOPLTXPVRDBG-UHFFFAOYSA-N 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
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- 102000018358 immunoglobulin Human genes 0.000 description 1
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- 229920002521 macromolecule Polymers 0.000 description 1
- 229910001425 magnesium ion Inorganic materials 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000000712 neurohormone Substances 0.000 description 1
- 238000004451 qualitative analysis Methods 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- RGHFKWPGWBFQLN-UHFFFAOYSA-M sodium;5,5-diethylpyrimidin-3-ide-2,4,6-trione Chemical compound [Na+].CCC1(CC)C([O-])=NC(=O)NC1=O RGHFKWPGWBFQLN-UHFFFAOYSA-M 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 239000010409 thin film Substances 0.000 description 1
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Images
Classifications
-
- 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
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D57/00—Separation, other than separation of solids, not fully covered by a single other group or subclass, e.g. B03C
- B01D57/02—Separation, other than separation of solids, not fully covered by a single other group or subclass, e.g. B03C by electrophoresis
Abstract
The invention provides an electrophoresis buffer solution which is prepared by adjusting the pH value to 8.3-8.6 by the following raw materials: tris, disodium EDTA, NaCl and distilled water. When the electrophoresis buffer solution prepared by the invention is used for carrying out a serum acetate fiber film electrophoresis experiment, the probability of five strips is high, the zone is clear and visible, and the zone is obvious; the buffer solution prepared by the invention has low ionic strength, is an alkaline buffer solution, is easy to adjust the pH value of the buffer solution, is easy to calculate the ionic strength, and is suitable for developing all electrophoresis experiments for replacing barbital-barbital sodium. In addition, the electrophoresis buffer solution prepared by the invention can be used in other life science experimental researches with different pH values and different ionic strengths according to requirements.
Description
Technical Field
The invention belongs to the technical field of biochemistry, and particularly relates to an electrophoresis buffer solution.
Background
Biochemistry is an important professional basic course of common college medicine, animal medicine and production profession, and is an experiment-based subject. The experimental teaching focuses on qualitative and quantitative analysis technology of macromolecular substances, wherein an important and basic experimental technology is electrophoresis.
The electrophoresis experiment especially uses the cellulose acetate film electrophoresis which is simple, convenient, visual and practical, has the characteristics of small electroosmosis, high separation speed, clear zone, simple operation, small sample dosage and the like, has very high development rate in colleges and universities, and is one of the most basic teaching items in the college biochemical experiment teaching. The method utilizes protein as ampholyte. Under a certain pH environment, the amount of charges on the surface of the protein is different, and the size and the form of the particles are different, namely under the condition of electrification, the higher the voltage in an electric field with different migration speeds in the electric field, the higher the acting force of the electrode on charged particles, the higher the migration speed of the particles is, but the voltage is also influenced by the length of a supporting medium, and the moving speed of the charged particles is in direct proportion to the voltage difference per unit length on the supporting medium, namely in direct proportion to the potential gradient, so that different proteins can be separated. In recent years, two medicines of barbital and barbital sodium are not easy to purchase, on one hand, the factors are that the control of special medicines is strict, and on the other hand, the required amount of experimental medicines is large and the purchase amount is limited. Moreover, barbiturate-barbiturate sodium buffer solution is easy to form crystals at two ends of an electrode in the experimental process to influence the electrophoresis efficiency, so that the positive result is difficult to judge.
The serum protein electrophoresis has wide application prospect, wherein the serum protein can be separated into five obvious zones of albumin, alpha 1-globulin, alpha 2-globulin, beta-globulin and gamma-globulin in the serum protein acetate thin film electrophoresis. Abnormal changes in certain serum proteins can be used to predict the occurrence of certain diseases, for example, serum globulin levels have certain effects on the levels of inflammatory factors and neurohormones in patients with acute ischemic stroke after neurointervention, and abnormal increase in serum globulin may also have certain relationship with the occurrence of diseases of multiple myeloma. Multiple myeloma secretes abnormal monoclonal immunoglobulin (M protein) which results in an increase in abnormal globulin, a decrease in normal globulin synthesis, and the like. Therefore, it is important to develop an electrophoresis buffer.
Disclosure of Invention
The technical problem to be solved by the invention is to provide an electrophoresis buffer solution aiming at the defects of the prior art, the electrophoresis buffer solution prepared by the invention has high probability of five zone results, NaCl is added, the generation probability of electroosmosis is effectively reduced, and meanwhile, the ionic strength of a buffer system is increased by adding NaCl, so that the electrophoresis experiment is more suitable.
In order to solve the technical problems, the invention adopts the technical scheme that: an electrophoresis buffer solution is prepared by adjusting the pH value to 8.3-8.6 by the following raw materials: tris (tris-base), disodium EDTA, NaCl, and distilled water.
Preferably, the dosage ratio of the tris (tris-base), the disodium EDTA, the NaCl and the distilled water is 10.800 g: 0.930 g: 1.317 g: 1L of the total amount of the active ingredients.
Preferably, the pH is adjusted using a nitric acid solution.
Preferably, the mass fraction of the nitric acid solution is 50%.
THE invention also provides a preparation method of THE electrophoresis buffer solution, which comprises THE steps of adding 10.800g of tris-hydroxymethyl aminomethane (tris-alkali), 0.930g of disodium EDTA and 1.317g of NaCl into 1L of distilled water for full dissolution to obtain a prepared solution, and adjusting THE pH value of THE prepared solution to 8.3-8.6 by using a nitric acid solution to obtain THE THE electrophoresis buffer solution.
THE invention also provides application of THE prepared THE electrophoresis buffer solution, and THE THE electrophoresis buffer solution is used in an electrophoresis experiment.
Compared with the prior art, the invention has the following advantages:
compared with THE traditional barbiturate sodium electrophoresis buffer solution, THE THE electrophoresis buffer solution prepared by THE method has THE advantages that THE zones are clearer and more orderly, THE separation is convenient, THE experiment success rate is high, THE probability of five zone results is high, NaCl is added, THE generation probability of electroosmosis is effectively reduced, and meanwhile, THE ionic strength of a buffer system is increased by adding NaCl, so that THE electrophoresis experiment is more suitable. The pH value of the aqueous solution of Tris alkali is about 10.5, a nitric acid reagent is added to adjust the pH value to a required value, so that a buffer solution with the pH value can be obtained, Tris alkali is easy to purchase as a common buffer solution, the amount of the used nitric acid is approximate to trace, EDTA disodium is added, the EDTA disodium is common and easy to purchase as a metal complexing agent, and magnesium ions and the like in serum are effectively complexed.
It is specifically noted that THE THE buffer is not limited to an electrophoresis experiment, and THE experiments such as agarose gel electrophoresis and nucleic acid electrophoresis are all to be studied.
The present invention will be described in further detail with reference to the accompanying drawings and examples.
Drawings
FIG. 1 shows THE results of THE experiments with THE THE running buffer prepared in example 2.
FIG. 2 shows the results of the experiment using the barbiturate-barbiturate sodium buffer in example 2.
FIG. 3 shows THE results of THE experiment with THE THE1 electrophoresis buffer prepared in comparative example 1.
FIG. 4 is the experimental results of the barbiturate-barbiturate sodium buffer in comparative example 1.
Detailed Description
Example 1
The electrophoresis buffer solution of the embodiment is prepared by adjusting the pH value to 8.3-8.6 by the following raw materials: tris (tris-base), disodium EDTA, NaCl, and distilled water.
The dosage ratio of the tris (tris-alkali), the EDTA disodium, the NaCl and the distilled water is 10.800 g: 0.930 g: 1.317 g: 1L; the pH is adjusted by using a nitric acid solution, and the mass fraction of the nitric acid solution is 50%.
The preparation method of the electrophoresis buffer solution comprises the following steps:
adding 10.800g of tris-base, 0.930g of disodium EDTA and 1.317g of NaCl into 1L of distilled water, and fully dissolving to obtain a prepared solution, and adjusting THE pH value of THE prepared solution to 8.3 by using a nitric acid solution to prepare THE THE electrophoresis buffer solution.
THE THE electrophoresis buffer prepared in this example was used for acetate membrane electrophoresis experiments.
1. Test materials:
equipment: an analytical balance, an electrophoresis apparatus, an electrophoresis tank, a sample applicator, an iron support (with an iron clamp), an acid burette (50ml), a plurality of beakers with different measuring ranges, a glass rod and a pH meter;
2. reagent:
(1) fresh serum (rabbit serum, no clotting phenomenon during manufacture);
(2) THE che running buffer prepared in this example;
(3) dyeing liquid: 0.5g of amino black 10B, adding 40ml of distilled water for dissolving, 50ml of methanol and 10ml of glacial acetic acid, fully mixing uniformly and storing in a reagent bottle;
(4) rinsing liquid: 45ml of 95 percent ethanol, 5ml of glacial acetic acid and 50ml of distilled water are mixed evenly;
(5) acetate film (2x8 cm).
3. Soaking
THE he electrophoresis buffer prepared in this example was poured into a small amount into a plate a, THE matte surfaces of 2 acetate films were identified, and a pencil was used as a mark, and at THE same time, a straight line perpendicular to THE length of THE film was drawn at a position 2cm from one end of THE matte surface with THE pencil to indicate THE spot application position and mark a1, a2, and then THE film was allowed to naturally sink and soaked for 30 minutes by dipping THE matte surface downward in THE electrophoresis buffer of plate a.
4. Spotting is carried out
The fully soaked membrane was removed from plate a, sandwiched between two layers of coarse filter paper to blott excess buffer, and then placed flat on a clean glass plate with the matte side up. One glass slide is taken, fresh serum is sucked on the glass slide by a20 ul pipette gun, the glass slide is inclined to enable the serum to be evenly dispersed, then a sample applicator is used for dipping the serum, and the sample is applied at the position with the pencil trace on one end of the film gently and straightly and then lifted. This spotted a thin strip of serum sample onto the membrane.
5. Electrophoresis
And (3) placing a film strip with a matt surface and a film strip with a sample a1 and a2, namely a sample application surface vertical filter paper bridge downwards on a filter paper bridge (four layers of filter paper are used as the bridge, the filter paper is completely wetted by buffer solution and bubbles are removed, so that the filter paper is tightly attached to a tank frame to form the filter paper bridge), tightly attaching the film and the filter paper, covering, electrifying after balancing for 10 minutes, adjusting the current intensity to be 20mA, controlling the voltage to be 70v, electrifying for 50 minutes, and closing the power supply.
6. Dyeing process
And after electrifying, taking out the film by using toothless forceps, directly soaking the film in a culture dish containing the amino black staining solution, and taking out the film after 5 minutes.
7. Rinsing
And (3) immersing the dyed film into a culture dish containing rinsing liquid, and repeatedly rinsing for 3 times to remove the background color.
The experiment is repeated for 10 times according to the steps to obtain 20 acetate films a1, a2, a3, a4, a5, a6, a7, a8, a9, a10, a11, a12, a13, a14, a15, a16, a17, a18, a19 and a20 in total.
8. Results of the experiment
TABLE 1 number of bands appearing in acetate fiber membrane electrophoresis experiment with THE THE electrophoresis buffer prepared in this example
| Acetate fiber film | a1 | a2 | a3 | a4 | a5 | a6 | a7 | a8 | a9 | a10 |
| Number of appearance zone | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 4 | 5 | 5 |
| Acetate fiber film | a11 | a12 | a13 | a14 | a15 | a16 | a17 | a18 | a19 | a20 |
| Number of appearance zone | 5 | 4 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 |
The results are shown in Table 1, which totals 20 times, wherein five of the results are 18 times and four of the results are 2 times.
The probability of five acetate fiber films is as follows:
P＝18/20*100％＝90％
therefore, THE success rate of THE experiment of THE THE electrophoresis buffer solution prepared by THE embodiment in THE serum protein acetate fiber film is 100%, and THE probability of five zones is 90%.
Example 2
The electrophoresis buffer solution of the embodiment is prepared by adjusting the pH value to 8.3-8.6 by the following raw materials: tris (tris-base), disodium EDTA, NaCl, and distilled water.
The dosage ratio of the tris (tris-alkali), the EDTA disodium, the NaCl and the distilled water is 10.800 g: 0.930 g: 1.317 g: 1L; the pH is adjusted by using a nitric acid solution, and the mass fraction of the nitric acid solution is 50%.
The preparation method of the electrophoresis buffer solution comprises the following steps:
adding 10.800g of tris-base, 0.930g of disodium EDTA and 1.317g of NaCl into 1L of distilled water to obtain a preparation solution, and adjusting THE pH value of THE preparation solution to 8.6 by using a nitric acid solution to prepare THE THE electrophoresis buffer solution.
THE THE electrophoresis buffer prepared in this example was compared with a barbiturate-barbiturate sodium buffer pH8.6 for cellulose acetate membrane electrophoresis experiments.
1. Test materials:
equipment: an analytical balance, an electrophoresis apparatus, an electrophoresis tank, a sample applicator, an iron support (with an iron clamp), an acid burette (50ml), a plurality of beakers with different measuring ranges, a glass rod and a pH meter;
2. reagent:
(1) fresh serum (rabbit serum, no clotting phenomenon during manufacture);
(2) THE che running buffer prepared in this example; barbital-barbital sodium electrophoresis buffer (pH 8.6);
(3) dyeing liquid: 0.5g of amino black 10B, adding 40ml of distilled water for dissolving, 50ml of methanol and 10ml of glacial acetic acid, fully mixing uniformly and storing in a reagent bottle;
(4) rinsing liquid: 45ml of 95 percent ethanol, 5ml of glacial acetic acid and 50ml of distilled water are mixed evenly;
(5) acetate film (2x8 cm).
3. Soaking
THE he electrophoresis buffer prepared in this example was poured into a small amount into a plate 1, THE matte surface of 1 acetate film was identified, and a pencil was used as a mark, and at THE same time, a straight line perpendicular to THE length of THE film was drawn at a position 2cm from one end of THE matte surface with THE pencil to indicate THE sample application position and mark No. 1, and then THE matte surface was lowered and immersed in THE electrophoresis buffer of THE plate 1 for 30 minutes.
Pouring a small amount of barbiturate-barbiturate sodium electrophoresis buffer solution into a plate 2, identifying the matt surface of 1 acetate fiber film, using a pencil as a mark, simultaneously using the pencil to make a straight line perpendicular to the length of the film at a position 2cm away from one end of the matt surface to represent the sample application position and mark the No. 2, then soaking the matt surface downwards into the electrophoresis buffer solution of the plate 2 to enable the film to naturally sink, and soaking for 30 minutes.
4. Spotting is carried out
The fully soaked membrane was removed from plates 1 and 2, sandwiched between two layers of coarse filter paper to remove excess buffer, and then placed flat on a clean glass plate with the matte side up. One glass slide is taken, fresh serum is sucked on the glass slide by a20 ul pipette gun, the glass slide is inclined to enable the serum to be evenly dispersed, then a sample applicator is used for dipping the serum, and the sample is applied at the position with the pencil trace on one end of the film gently and straightly and then lifted. This spotted a thin strip of serum sample onto the membrane.
5. Electrophoresis
No. 1 and No. 2 film strips with samples have no gloss surfaces, namely sample application surfaces are vertical to a filter paper bridge and are placed downwards on the filter paper bridge of an electrophoresis tank (four layers of filter paper are used as the bridge, the filter paper is completely wetted by buffer solution and bubbles are removed, so that the filter paper is tightly attached to a tank frame to form the filter paper bridge), and the film and the filter paper need to be tightly attached. And covering the cover, electrifying after balancing for 10 minutes, adjusting the current intensity to be 20mA, the voltage to be 70v, electrifying for 50 minutes, and closing the power supply.
6. Dyeing process
And after electrifying, taking out the film by using toothless forceps, directly soaking the film in a culture dish containing the amino black staining solution, and taking out the film after 5 minutes.
7. Rinsing
And (3) immersing the dyed film into a culture dish containing rinsing liquid, and repeatedly rinsing for 3 times to remove the background color.
The experiment is repeated for 10 times according to the steps to obtain 20 acetate films b1, b2, b3, b4, b5, b6, b7, b8, b9, b10, b11, b12, b13, b14, b15, b16, b17, b18, b19 and b20 in total.
8. Results of the experiment
As shown in FIG. 1, THE experimental results of THE THE electrophoresis buffer prepared in this example (No. 1): clear 5 zones can be presented on the film, and albumin, alpha 1-globulin, alpha 2-globulin, beta-globulin and gamma-globulin are sequentially arranged from the positive terminal.
As shown in fig. 2, the results of the barbiturate-barbiturate sodium buffer experiment: (No. 2): clear 4 zones can be presented on the film, and albumin, alpha 1-globulin, alpha 2, beta-globulin and gamma-globulin are sequentially arranged from the positive terminal.
Comparative example 1
The electrophoresis buffer of the comparative example was prepared by adjusting the pH to 8.3-8.6 with the following raw materials: tris (hydroxymethyl) aminomethane (tris-base), disodium EDTA, and distilled water.
The dosage ratio of the tris (tris-alkali), the EDTA disodium and the distilled water is 10.800 g: 0.930 g: 1L; the pH is adjusted by using a nitric acid solution, and the mass fraction of the nitric acid solution is 50%.
The preparation method of the electrophoresis buffer solution of the comparative example comprises the following steps:
adding 10.800g of tris-base and 0.930g of disodium EDTA into 1L of distilled water to obtain a preparation solution, and adjusting THE pH value of THE preparation solution to 8.6 by using a nitric acid solution to obtain THE THE1 electrophoresis buffer solution.
THE THE1 electrophoresis buffer solution prepared by THE comparative example and THE barbiturate-barbiturate sodium buffer solution with THE pH of 8.6 are used for carrying out THE comparison of THE cellulose acetate film electrophoresis experiment.
1. Test materials:
equipment: an analytical balance, an electrophoresis apparatus, an electrophoresis tank, a sample applicator, an iron support (with an iron clamp), an acid burette (50ml), a plurality of beakers with different measuring ranges, a glass rod and a pH meter;
2. reagent:
(1) fresh serum (rabbit serum, no clotting phenomenon during manufacture);
(2) THE THE1 electrophoresis buffer prepared in this comparative example; barbital-barbital sodium electrophoresis buffer (pH 8.6);
(3) dyeing liquid: 0.5g of amino black 10B, adding 40ml of distilled water for dissolving, 50ml of methanol and 10ml of glacial acetic acid, fully mixing uniformly and storing in a reagent bottle;
(4) rinsing liquid: 45ml of 95 percent ethanol, 5ml of glacial acetic acid and 50ml of distilled water are mixed evenly;
(5) acetate film (2x8 cm).
3. Soaking
THE THE1 electrophoresis buffer solution prepared in this comparative example was poured into a small amount into a plate 3, then THE matte surface of 1 acetate film was identified, and a pencil was used as a mark, and at THE same time, a straight line perpendicular to THE length of THE film was made at a position 2cm from one end of THE matte surface with THE pencil to indicate THE sample application position and mark No. 3, and then THE film was allowed to sink naturally by immersing THE matte surface downward in THE electrophoresis buffer solution in THE plate 3 for 30 minutes.
Pouring a small amount of barbiturate-barbiturate sodium electrophoresis buffer solution into a plate 4, identifying the matt surface of 1 acetate fiber film, using a pencil as a mark, simultaneously using the pencil to make a straight line perpendicular to the length of the film at a position 2cm away from one end of the matt surface to represent the sample application position and mark the No. 4, then soaking the matt surface downwards into the electrophoresis buffer solution of the plate 4 to enable the film to naturally sink, and soaking for 30 minutes.
4. Spotting is carried out
The fully soaked membrane was removed from plates 3, 4, sandwiched between two layers of coarse filter paper to remove excess buffer, and then placed flat on a clean glass plate with the matte side up. One glass slide is taken, fresh serum is sucked on the glass slide by a20 ul pipette gun, the glass slide is inclined to enable the serum to be evenly dispersed, then a sample applicator is used for dipping the serum, and the sample is applied at the position with the pencil trace on one end of the film gently and straightly and then lifted. This spotted a thin strip of serum sample onto the membrane.
5. Electrophoresis
No. 3 and No. 4 and No. 2 film strips with samples are placed on a filter paper bridge (four layers of filter paper are used as the bridge, the filter paper is completely wetted by buffer solution and bubbles are removed, so that the filter paper is tightly attached to a tank frame to form the filter paper bridge) of an electrophoresis tank, wherein the matt surface, namely the sample application surface, of each film strip is vertical to the filter paper bridge, and the film and the filter paper are required to be tightly attached. And covering the cover, electrifying after balancing for 10 minutes, adjusting the current intensity to be 20mA, the voltage to be 70v, electrifying for 50 minutes, and closing the power supply.
6. Dyeing process
And after electrifying, taking out the film by using toothless forceps, directly soaking the film in a culture dish containing the amino black staining solution, and taking out the film after 5 minutes.
7. Rinsing
And (3) immersing the dyed film into a culture dish containing rinsing liquid, and repeatedly rinsing for 3 times to remove the background color.
The experiment is repeated for 10 times according to the steps to obtain 20 acetate films c1, c2, c3, c4, c5, c6, c7, c8, c9, c10, c11, c12, c13, c14, c15, c16, c17, c18, c19 and c20 in total.
8. Results of the experiment
As shown in fig. 3, THE results of THE experiment of THE che 1 running buffer prepared in this comparative example (No. 3): clear 5 zones can be presented on the film, and albumin, alpha 1-globulin, alpha 2-globulin, beta-globulin and gamma-globulin are sequentially arranged from the positive terminal.
As shown in fig. 4, the results of the barbiturate-barbiturate sodium buffer experiment: (No. 4): clear 4 zones can be presented on the film, and albumin, alpha 1-globulin, alpha 2, beta-globulin and gamma-globulin are sequentially arranged from the positive terminal.
In summary, it can be found that THE experimental results of THE che electrophoresis buffer solution prepared in example 2 are better than those of THE che 1 electrophoresis buffer solution and THE barbital-barbital sodium buffer solution prepared in comparative example 1, NaCl is not added to THE che 1 electrophoresis buffer solution prepared in comparative example 1, and NaCl is added to THE che electrophoresis buffer solution prepared in example 2, so that THE generation probability of THE electroosmosis phenomenon is effectively reduced, and meanwhile, THE ionic strength of THE buffer system is increased by adding NaCl, so that THE electrophoresis experiment is more suitable.
Example 3
The electrophoresis buffer solution of the embodiment is prepared by adjusting the pH value to 8.3-8.6 by the following raw materials: tris (tris-base), disodium EDTA, NaCl, and distilled water.
The dosage ratio of the tris (tris-alkali), the EDTA disodium, the NaCl and the distilled water is 10.800 g: 0.930 g: 1.317 g: 1L; the pH is adjusted by using a nitric acid solution, and the mass fraction of the nitric acid solution is 50%.
The preparation method of the electrophoresis buffer solution comprises the following steps:
adding 10.800g of tris-base, 0.930g of disodium EDTA and 1.317g of NaCl into 1L of distilled water, and fully dissolving to obtain a prepared solution, and adjusting THE pH value of THE prepared solution to 8.4 by using a nitric acid solution to prepare THE THE electrophoresis buffer solution.
The above description is only for the preferred embodiment of the present invention, and is not intended to limit the present invention in any way. Any simple modification, change and equivalent changes of the above embodiments according to the technical essence of the invention are still within the protection scope of the technical solution of the invention.
Claims (4)
1. An electrophoresis buffer solution is characterized by being prepared by adjusting the pH value to 8.3-8.6 by the following raw materials: tris, disodium EDTA, NaCl and distilled water.
2. The running buffer of claim 1, wherein the tris, disodium EDTA, NaCl, and distilled water are used in a ratio of 10.800 g: 0.930 g: 1.317 g: 1L of the compound.
3. An electrophoresis buffer according to claim 1 or 2 wherein the pH is adjusted using a nitric acid solution.
4. An electrophoresis buffer according to claim 3 wherein the nitric acid solution is present in an amount of 50% by weight.
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