CN108627564A - A kind of polyacrylamide gel of efficient stable and preparation method thereof - Google Patents
A kind of polyacrylamide gel of efficient stable and preparation method thereof Download PDFInfo
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- CN108627564A CN108627564A CN201810433719.2A CN201810433719A CN108627564A CN 108627564 A CN108627564 A CN 108627564A CN 201810433719 A CN201810433719 A CN 201810433719A CN 108627564 A CN108627564 A CN 108627564A
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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
- G01N27/44704—Details; Accessories
- G01N27/44747—Composition of gel or of carrier mixture
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/40—Concentrating samples
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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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/40—Concentrating samples
- G01N2001/4038—Concentrating samples electric methods, e.g. electromigration, electrophoresis, ionisation
Abstract
The invention discloses a kind of polyacrylamide gel of efficient stable and preparation method thereof, which is made of the separation gel comprising acrylamide, double methene acrylamides, glycine, pH stabilizers HEPES, lauryl sodium sulfate, taurine, ultra-pure water, Tris HCl solutions, ammonium persulfate solution and tetramethylethylenediamine and concentration glue.The present invention successfully improves the sharpness and stability of polyacrylamide gel electrophoresis band, can be preserved at ambient temperature up to year, not influence the effect of protein electrophoresis by the way that the reagents such as glycine are added.Meanwhile product of the invention can individually need not buy expensive matched reagent again with the Eco-power TRIS Glycine systems of compatible conventional, use cost is cheaper.The product of the present invention can complete voltage under conditions of high voltage, and highest can reach 200V voltages, can complete to test at 30 minutes, shorten the time of electrophoresis.
Description
Technical field
The invention belongs to biological medicine and Scientific Research Service fields, and in particular to a kind of polyacrylamide gel of efficient stable
And preparation method thereof.
Background technology
Polyacrylamide gel electrophoresis, polyacrylamide gel electrophoresis, abbreviation PAGE are with poly-
A kind of common electrophoretic techniques of the acrylamide gel as supporting dielectric is used for protein isolate matter.Polyacrylamide gel is net
Shape structure has molecular sieving effect.There are two types of forms for it：Native polyacrylamide gel electrophoresis（Native-PAGE）With
SDS- polyacrylamide gels（SDS-PAGE）.Non-denaturing polyacrylamide gel, during electrophoresis, protein can be protected
Good working condition is held, and is in gradually ladder according to the molecular size range of protein, the shape of protein and its incidental quantity of electric charge
Degree separates.And SDS-PAGE can separating albumen matter according only to the difference of protein subunit molecular weight.The technology initially by
Shapiro was established in 1967, they have found that zwitterionic detergent and strong reduction are added in sample media and acrylamide gel
Agent（SDS, that is, lauryl sodium sulfate）Afterwards, the electrophoretic mobility of protein subunit depends primarily on the size of molecular weight subunit
（Charge factor can be ignored）.Polyacrylamide gel is polymerized by monomeric acrylamide and methylene diacrylamide, polymerization
Process is catalyzed by free radical and is completed.There are two types of the common methods of catalytic polymerization：Chemical polymerization and light polymerization method.Chemical polymerization with
Ammonium persulfate（APS）For catalyst, with tetramethylethylenediamine（TEMED）For accelerator.In the course of the polymerization process, TEMED was catalyzed
Ammonium sulfate generates free radicals, and the latter causes acrylamide monomer polymerization, while methylene diacrylamide is produced with acrylamide interchain
Raw methene key crosslinking, to form tridimensional network.
According to polyacrylamide gel electrophoresis systems whether there is or not concentration effect, it is divided into continuous system and discontinuous system two is big
Class.PH of cushioning fluid and gel strength are identical in continuous system electrophoretic system, and charged particle is under electric field action, mainly by charge
And molecular sieving effect.Due to buffer solution ion component in discontinuous system, pH, the discontinuity of gel strength and electric potential gradient,
Swimming not only has charge effect, molecular sieving effect also to have concentration effect to charged particle in the electric field, thus its separated bands is clear
Clear degree and resolution ratio are good compared with the former.Discontinuous system is made of electrode buffer, concentration glue and separation gel.Concentration glue be by
Large-pore gel made of AP catalytic polymerizations, gel buffer liquid are the Tris-HCl of pH6.7.Separation gel is made of AP catalytic polymerizations
Aperture glue, gel buffer liquid are pH8.9 Tris-HCl.Electrode buffer is pH8.3 Tris- glycine buffers.2 kinds of apertures
Gel, 2 kinds of buffer systems, 3 kinds of pH value so that discontinuous system is formd gel aperture, pH value, buffer solution ion component not
Continuity, this is the principal element of sample concentration.
Immunoblotting（Western blot test）, i.e. Western Blot are a kind of the most frequently used in Protein Detection and most
One of important method, be by electrophoretic separation after cell or tissue gross protein from gel be transferred to solid support NC films or
On pvdf membrane, a kind of protein detection techniques of detection of specific antibody specific antigen are then used, base has been widely used in
Because in many aspects such as the expression study of protein level, antibody activity detection and disease early diagnosis.Entire Western blotting is real
It is one continuous to test, and systematic process, complete step is related to as follows：1. the sample preparation of protein;2. protein is determined
Amount；3.PAGE electrophoresis；4. transferring film；5. blocking non-specific；6. immuning hybridization reacts；7. chemiluminescence developing and fixing.
Wherein each step is critically important to final result, does not only make mistakes in each experiment of progress, just can guarantee acquisition
Expected result.Wherein each experimental procedure can need a series of reagent auxiliary that could complete, therefore, the examination being simple and efficient
Agent is essential.
PAGE electrophoretic systems are made of electrode buffer, concentration glue and separation gel.It is by ammonium persulfate and four to concentrate glue
Large-pore gel made of methyl ethylenediamine catalytic polymerization, gel buffer liquid are the Tris-HCl of pH6.8.Separation gel is poly- by AP catalysis
Aperture glue made of conjunction, gel buffer liquid are the Tris-HCl of pH8.9.Electrode buffer is that the Tris- glycine of pH8.3 is slow
Fliud flushing.Conventional preparation PAGE glue is by acrylamide, methylene diacrylamide, Tris-HCl, ammonium persulfate, SDS, tetramethyl
Ethylenediamine and distilled water configure according to a certain percentage, first to configure separation gel it is to be solidified and then configuration concentration glue, plug comb
Son just completes PAGE glue preparation process.The process will calculate certain ratio with glue, and gel sets is waited for need the time, in addition by
It is to preserve for a long time in the characteristics of conventional gel, is generally finished within one week, therefore causes often to configure PAGE
Glue, this needs a large amount of time and efforts, is unfavorable for the quick progress of albumen research.
Although Ye You companies have developed the PAGE pre-prepared colloid products that can be preserved for a long time currently on the market, they
Product is required to specific buffer solution system, such as MOPS buffer solutions, and with conventional Eco-power TRIS-Glycine systems
It can not be compatible with, therefore cause the cost used very high, also be unfavorable for widely promoting the use of.
Invention content
The shortcomings that for above-mentioned conventional PAGE glue, the present invention is intended to provide a kind of polyacrylamide gel of efficient stable and
Preparation method.
To realize above-mentioned technical purpose and the technique effect, the invention is realized by the following technical scheme：
A kind of polyacrylamide gel of efficient stable is composed of separation gel and concentration glue；
The component for including in the separation gel has acrylamide, double methene acrylamides, glycine, pH stabilizers HEPES, 12
Sodium alkyl sulfate, taurine, ultra-pure water, Tris-HCl solution, ammonium persulfate solution and tetramethylethylenediamine；
Component included in the concentration glue has acrylamide, double methene acrylamides, glycine, pH stabilizers HEPES, ten
Sodium dialkyl sulfate, taurine, ultra-pure water, Tris-HCl solution, ammonium persulfate solution and tetramethylethylenediamine.
Further, in the separation gel, the quality of acrylamide is 80-120g, and the quality of double methene acrylamides is
The quality of 2.4-3.6g, glycine are 25g, and the quality of pH stabilizers HEPES is 5g, and the quality of lauryl sodium sulfate is
The quality of 0.5g, taurine are 10g, and the volume of ultra-pure water is 600mL, and the volume of Tris-HCl solution is 100mL, persulfuric acid
The volume of ammonium salt solution is 10mL, tetramethylethylenediamine 0.5mL.
Further, in the concentration glue, the quality of acrylamide is 50g, and the quality of double methene acrylamides is 1.5g,
The quality of glycine is 25g, and the quality of pH stabilizers HEPES is 5g, and the quality of lauryl sodium sulfate is 0.5g, taurine
Quality is 10g, and the volume of ultra-pure water is 600mL, and the volume of Tris-HCl solution is 100mL, the volume of ammonium persulfate solution
For 10mL, tetramethylethylenediamine 0.5mL.
Further, the Tris-HCl solution in the separation gel and the concentration glue is the Tris-HCl of 1M pH6.5
Solution.
Further, the ammonium persulfate solution in the separation gel and the concentration glue is 10% ammonium persulfate solution.
A kind of preparation method of the polyacrylamide gel of efficient stable, includes the following steps：
Step 1）Weigh a certain amount of acrylamide, double methene acrylamides, glycine, pH stabilizers HEPES, dodecyl sulphur
Sour sodium, taurine are placed in 1000ml beakers；
Step 2）A certain amount of ultra-pure water is added in rapid beaker one step up, is sufficiently stirred mixing, forms mixed solution；
Step 3）In previous step obtains mixed solution, a certain amount of Tris-HCl solution, ammonium persulfate solution and four is added
Methyl ethylenediamine is sufficiently stirred mixing, is configured to separation sol solution；
Step 4）Previous step is obtained separation sol solution to be added in the vertical glue glass plate of 1.0mm, is added suitable super
Pure water carries out crimping, and standing certain time waits for its solidification, forms separation gel；
Step 5）A certain amount of acrylamide, double methene acrylamides, glycine, pH stabilizers HEPES, dodecane are weighed again
Base sodium sulphate, taurine are placed in another 1000ml beaker；
Step 6）A certain amount of ultra-pure water is added in rapid beaker one step up, is sufficiently stirred mixing, forms mixed solution；
Step 7）In previous step obtains mixed solution, a certain amount of Tris-HCl solution, ammonium persulfate solution and four is added
Methyl ethylenediamine is sufficiently stirred mixing, is configured to concentration sol solution；
Step 8）The ultra-pure water for removing crimping above separation gel is used in combination blotting paper to blot residual liquid, and it is rigid that previous step is added
The concentration sol solution prepared is inserted into comb, stands certain time waiting gel, polyacrylamide gel is made.
Further, step 1）In, weigh 80-120g acrylamides, the bis- methene acrylamides of 2.4-3.6g, the sweet ammonia of 25g
Acid, 5gpH stabilizers HEPES, 0.5g lauryl sodium sulfate, 10g taurines.
Further, step 2）With step 6）In, the ultra-pure water of 600mL is added.
Further, step 3）With step 7）In, 1M pH6.5 Tris-HCl solution, the 10ml of the 100ml being added
10% ammonium persulfate solution and the tetramethylethylenediamine of 1ml.
Further, the step 4）With step 8）In, gel time of repose is 20-30 minutes.
The polyacrylamide gel of the present invention may be implemented to preserve for a long time：In conventional polypropylene acrylamide gel system,
Separation gel is the aperture glue made of APS and TEMED catalytic polymerizations, and gel buffer liquid is pH8.9 Tris-HCl, buffer electrode
Liquid is pH8.3 Tris- glycine buffers.But acrylamide is unstable under conditions of pH value is more than 7, more than one
Preservation more than week is easy for leading to the variation in the aperture of gel, loses the effect of molecular sieve.If can be led for protein electrophoresis
The resolution ratio of protein is caused to reduce, band is fuzzy, is no longer available for the detection of albumen.By we have discovered that, pH be less than 7
Gel buffer liquid in suitable glycine is added can stablize the aperture of acrylamide, while the examinations such as pH stabilizers HEPES are added
Agent may be implemented the stabilization for preserving polyacrylamide gel for a long time, not interfere with the variation of the resolution ratio of protein.
The polyacrylamide gel of the present invention can be compatible with common TRIS-Glycine electrophoretic systems：Polyacrylamide is solidifying
Gel electrophoresis buffer solution is there are many system, such as MOPS electrophoresis liquids, MES electrophoresis liquids, and TRIS-Glycine electrophoresis liquids etc., but it is most common
Be TRIS-Glycine electrophoresis liquids.Because TRIS and Glycine are common reagents, less expensive uses them
It is relatively low as buffer solution cost, save valuable reasearch funds.Existing partial precast glue product is required for mating in the market
Unique electrophoretic buffer cannot perfectly be compatible with TRIS-Glycine electrophoresis liquids.We have found both sexes by a large amount of experiment
Substance can play the role of electrophoresis ionic equilibrium, be added in pre-prepared colloid, while generate the synergistic effect of cation and anion,
So that coordinating the zwitterionic interaction of amino acid when albumen ionization, the function of the molecular sieve of polyacrylamide is realized, it is right
Protein realizes separation according to the size of fractional dose.
Beneficial effects of the present invention are as follows：
The present invention successfully improves the sharpness and stabilization of polyacrylamide gel electrophoresis band by the way that the reagents such as glycine are added
Property, it can be preserved at ambient temperature up to year, not influence the effect of protein electrophoresis.Meanwhile of the invention poly- third
Acrylamide gel can individually need not buy expensive mating examination again with the Eco-power TRIS-Glycine systems of compatible conventional
Agent, use cost are cheaper.The polyacrylamide gel of the present invention can complete voltage under conditions of high voltage, and highest can
To reach 200V voltages, it can complete to test at 30 minutes, shorten the time of electrophoresis.
Above description is only the general introduction of technical solution of the present invention, in order to better understand the technical means of the present invention,
And can be implemented in accordance with the contents of the specification, below with presently preferred embodiments of the present invention and after being described in detail such as.The present invention's
Specific implementation mode is shown in detail by following embodiment.
Specific implementation mode
Below with reference to specific embodiment, next the present invention will be described in detail.
Case study on implementation 1：Prepare the polyacrylamide gel of concentration 10%
（1）Configuration separation sol solution, weighs following reagent, is placed in 1000ml beakers：
Acrylamide 100g, double methene acrylamide 3g, glycine 25g, HEPES 5g, SDS 0.5g, taurine 10g；
（2）The ultra-pure water of about 600ml is added into beaker, is sufficiently stirred mixing；
（3）In above-mentioned solution, addition 100ml, 1M pH6.5 Tris-HCl solution, the ammonium persulfate solution of 10ml 10%,
The TEMED of 0.5ml, is sufficiently stirred mixing；
（4）Above-mentioned solution is added in the vertical glue glass plate of 1.0mm, suitable ultra-pure water is added and carries out crimping, stands
Wait for its solidification within 20-30 minutes；
（5）Configuration concentration sol solution, weighs following reagent, is placed in 1000ml beakers：
Acrylamide 50g, double methene acrylamide 1.5g, glycine 25g, HEPES 5g, SDS 0.5g, taurine 10g；
（6）The ultra-pure water of about 600ml is added into beaker, is sufficiently stirred mixing
（7）In above-mentioned solution, addition 100ml, 1M pH6.5 Tris-HCl solution, the ammonium persulfate solution of 10ml 10%,
The TEMED of 1ml, is sufficiently stirred mixing
（8）The ultra-pure water for removing above-mentioned crimping blots residual liquid with blotting paper, the concentration sol solution just prepared is added, so
After be inserted into comb, stand 20-30 minute waiting gels.
Case study on implementation 2：Prepare the polyacrylamide gel of concentration 8%
（1）Configuration separation sol solution, weighs following reagent, is placed in 1000ml beakers：
Acrylamide 80g, double methene acrylamide 2.4g, glycine 25g, HEPES 5g, SDS 0.5g, taurine
10g；
（2）The ultra-pure water of about 600ml is added into beaker, is sufficiently stirred mixing；
（3）In above-mentioned solution, addition 100ml, 1M pH6.5 Tris-HCl solution, the ammonium persulfate solution of 10ml 10%,
The TEMED of 0.5ml, is sufficiently stirred mixing；
（4）Above-mentioned solution is added in the vertical glue glass plate of 1.0mm, suitable ultra-pure water is added and carries out crimping, stands
Wait for its solidification within 20-30 minutes；
（5）Configuration concentration sol solution, weighs following reagent, is placed in 1000ml beakers：
Acrylamide 50g, double methene acrylamide 1.5g, glycine 25g, HEPES 5g, SDS 0.5g, taurine 10g；
（6）The ultra-pure water of about 600ml is added into beaker, is sufficiently stirred mixing
（7）In above-mentioned solution, addition 100ml, 1M pH6.5 Tris-HCl solution, the ammonium persulfate solution of 10ml 10%,
The TEMED of 1ml, is sufficiently stirred mixing
（8）The ultra-pure water for removing above-mentioned crimping blots residual liquid with blotting paper, the concentration sol solution just prepared is added, so
After be inserted into comb, stand 20-30 minute waiting gels.
Case study on implementation 3：Prepare the polyacrylamide gel of concentration 12%
（1）Configuration separation sol solution, weighs following reagent, is placed in 1000ml beakers：
Acrylamide 120g, double methene acrylamide 3.6g, glycine 25g, HEPES 5g, SDS 0.5g, taurine
10g；
（2）The ultra-pure water of about 600ml is added into beaker, is sufficiently stirred mixing；
（3）In above-mentioned solution, addition 100ml, 1M pH6.5 Tris-HCl solution, the ammonium persulfate solution of 10ml 10%,
The TEMED of 0.5ml, is sufficiently stirred mixing；
（4）Above-mentioned solution is added in the vertical glue glass plate of 1.0mm, suitable ultra-pure water is added and carries out crimping, stands
Wait for its solidification within 20-30 minutes；
（5）Configuration concentration sol solution, weighs following reagent, is placed in 1000ml beakers：
Acrylamide 50g, double methene acrylamide 1.5g, glycine 25g, HEPES 5g, SDS 0.5g, taurine 10g；
（6）The ultra-pure water of about 600ml is added into beaker, is sufficiently stirred mixing
（7）In above-mentioned solution, addition 100ml, 1M pH6.5 Tris-HCl solution, the ammonium persulfate solution of 10ml 10%,
The TEMED of 1ml, is sufficiently stirred mixing
（8）The ultra-pure water for removing above-mentioned crimping blots residual liquid with blotting paper, the concentration sol solution just prepared is added, so
After be inserted into comb, stand 20-30 minute waiting gels.
Case study on implementation 4：Protein electrophoresis
Using the preparation of above-mentioned case study on implementation 1-3 various concentration poly- propionamide gel carry out electrophoresis experiment, by BSA samples into
Sample-loading buffer is added in row gradient dilution, and then loading that sample is added separately to above-mentioned gel is aerial, electrophoretic buffer
It uses Tris-Glycine electrophoretic systems, voltage to be set in 200V, while being added to PageRule Prestained
Protein Ladder are as measurement standard product.Electrophoresis time is set as 30 minutes, after electrophoresis finishes, by gel take out into
Row Germicidal efficacy.
Case study on implementation 5：Accelerated stability test
The polyacrylamide gel prepared in above-mentioned case study on implementation 1 is respectively placed under room temperature, separately sampled 3 days, 7
It, 11 days, is used as sample in 15 days, then carries out electrophoresis experiment.BSA samples are subjected to gradient dilution, sample-loading buffer are added, so
The loading that sample is added separately to above-mentioned gel afterwards is aerial, and electrophoretic buffer uses Tris-Glycine electrophoresis bodies
System, voltage is set in 200V, while being added to PageRule Prestained Protein Ladder as measurement standard
Product.Electrophoresis time is set as 30 minutes, after electrophoresis finishes, and gel is taken out and carries out Germicidal efficacy.
Certain examples detailed above made by the technical concepts and features of the present invention simply to illustrate that enumerate and non exhaustive, mesh
Be allow person skilled in the art to can understand the content of the present invention and implement it accordingly, the present invention can not be limited with this
Protection domain.According to the modification that the Spirit Essence of main skill scheme of the invention is done, all cover in protection scope of the present invention
Within.
Claims (10)
1. a kind of polyacrylamide gel of efficient stable, it is characterised in that：It is composed of separation gel and concentration glue；
The component for including in the separation gel has acrylamide, double methene acrylamides, glycine, pH stabilizers HEPES, 12
Sodium alkyl sulfate, taurine, ultra-pure water, Tris-HCl solution, ammonium persulfate solution and tetramethylethylenediamine；
Component included in the concentration glue has acrylamide, double methene acrylamides, glycine, pH stabilizers HEPES, ten
Sodium dialkyl sulfate, taurine, ultra-pure water, Tris-HCl solution, ammonium persulfate solution and tetramethylethylenediamine.
2. the polyacrylamide gel of efficient stable according to claim 1, it is characterised in that：In the separation gel, third
The quality of acrylamide is 80-120g, and the quality of double methene acrylamides is 2.4-3.6g, and the quality of glycine is 25g, and pH stablizes
The quality of agent HEPES is 5g, and the quality of lauryl sodium sulfate is 0.5g, and the quality of taurine is 10g, the volume of ultra-pure water
Volume for 600mL, Tris-HCl solution is 100mL, and the volume of ammonium persulfate solution is 10mL, tetramethylethylenediamine 0.5mL.
3. the polyacrylamide gel of efficient stable according to claim 1, it is characterised in that：In the concentration glue, third
The quality of acrylamide is 50g, and the quality of double methene acrylamides is 1.5g, and the quality of glycine is 25g, pH stabilizers HEPES
Quality be 5g, the quality of lauryl sodium sulfate is 0.5g, and the quality of taurine is 10g, and the volume of ultra-pure water is
The volume of 600mL, Tris-HCl solution is 100mL, and the volume of ammonium persulfate solution is 10mL, tetramethylethylenediamine 0.5mL.
4. the polyacrylamide gel of efficient stable according to claim 2 or 3, it is characterised in that：The separation gel and
Tris-HCl solution in the concentration glue is the Tris-HCl solution of 1M pH6.5.
5. the polyacrylamide gel of efficient stable according to claim 2 or 3, it is characterised in that：The separation gel and
Ammonium persulfate solution in the concentration glue is 10% ammonium persulfate solution.
6. a kind of preparation method of the polyacrylamide gel of efficient stable, which is characterized in that include the following steps：
Step 1）Weigh a certain amount of acrylamide, double methene acrylamides, glycine, pH stabilizers HEPES, dodecyl sulphur
Sour sodium, taurine are placed in 1000ml beakers；
Step 2）A certain amount of ultra-pure water is added in rapid beaker one step up, is sufficiently stirred mixing, forms mixed solution；
Step 3）In previous step obtains mixed solution, a certain amount of Tris-HCl solution, ammonium persulfate solution and four is added
Methyl ethylenediamine is sufficiently stirred mixing, is configured to separation sol solution；
Step 4）Previous step is obtained separation sol solution to be added in the vertical glue glass plate of 1.0mm, is added suitable super
Pure water carries out crimping, and standing certain time waits for its solidification, forms separation gel；
Step 5）A certain amount of acrylamide, double methene acrylamides, glycine, pH stabilizers HEPES, dodecane are weighed again
Base sodium sulphate, taurine are placed in another 1000ml beaker；
Step 6）A certain amount of ultra-pure water is added in rapid beaker one step up, is sufficiently stirred mixing, forms mixed solution；
Step 7）In previous step obtains mixed solution, a certain amount of Tris-HCl solution, ammonium persulfate solution and four is added
Methyl ethylenediamine is sufficiently stirred mixing, is configured to concentration sol solution；
Step 8）The ultra-pure water for removing crimping above separation gel is used in combination blotting paper to blot residual liquid, and it is rigid that previous step is added
The concentration sol solution prepared is inserted into comb, stands certain time waiting gel, polyacrylamide gel is made.
7. the preparation method of the polyacrylamide gel of efficient stable according to claim 6, it is characterised in that：Step 1）
In, weigh 80-120g acrylamides, the bis- methene acrylamides of 2.4-3.6g, 25g glycine, 5gpH stabilizers HEPES, 0.5g
Lauryl sodium sulfate, 10g taurines.
8. the preparation method of the polyacrylamide gel of efficient stable according to claim 6, it is characterised in that：Step 2）
With step 6）In, the ultra-pure water of 600mL is added.
9. the preparation method of the polyacrylamide gel of efficient stable according to claim 6, it is characterised in that：Step 3）
With step 7）In, the 1M pH6.5 Tris-HCl solution for the 100ml being added, the ammonium persulfate solution of 10ml 10% and 1ml
Tetramethylethylenediamine.
10. the preparation method of the polyacrylamide gel of efficient stable according to claim 6, it is characterised in that：It is described
Step 4）With step 8）In, gel time of repose is 20-30 minutes.
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Cited By (2)
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| CN109351293A (en) * | 2018-12-07 | 2019-02-19 | 陕西帆昌生物科技有限公司 | A kind of SDS-PAGE matches adhesive dispenser and its application method |
| CN112444552A (en) * | 2020-10-29 | 2021-03-05 | 江苏凯基生物技术股份有限公司 | Color-changeable polyacrylamide gel and kit containing same |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109351293A (en) * | 2018-12-07 | 2019-02-19 | 陕西帆昌生物科技有限公司 | A kind of SDS-PAGE matches adhesive dispenser and its application method |
| CN109351293B (en) * | 2018-12-07 | 2023-11-21 | 陕西帆昌生物科技有限公司 | SDS-PAGE glue distribution device and application method thereof |
| CN112444552A (en) * | 2020-10-29 | 2021-03-05 | 江苏凯基生物技术股份有限公司 | Color-changeable polyacrylamide gel and kit containing same |
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