CN107727724A - One kind detection method of protein - Google Patents
One kind detection method of protein Download PDFInfo
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- CN107727724A CN107727724A CN201711005742.3A CN201711005742A CN107727724A CN 107727724 A CN107727724 A CN 107727724A CN 201711005742 A CN201711005742 A CN 201711005742A CN 107727724 A CN107727724 A CN 107727724A
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- 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
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- G01N27/447—Systems using electrophoresis
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- G—PHYSICS
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Abstract
The invention discloses one kind to detect method of protein, comprises the following steps：(1) protein example is subjected to denaturation treatment, solution of dansyl chloride is then added into the protein example after denaturation treatment, 20 120min are placed at 30 50 DEG C；(2) SDS PAGE are prepared, the protein example added after step (1) processing, carry out gel electrophoresis operation；(3) after the completion of electrophoresis, protein adhesive is placed under uviol lamp or LED and observed, immediately arrives at testing result.The present invention carries out fluorescence labeling processing using dansyl Cl to protein example, sample progress synchronous with SDS PAGE preparation is handled, the step of need not be dyed and be decolourized again after electrophoresis, compared with existing detection method, experiment duration is substantially reduced, reduces experiment complexity.
Description
Technical field
The present invention relates to technical field of protein detection, and in particular to a kind of new detection method of protein.
Background technology
In life science field, it be unable to do without to the base substance protein of life and DNA research, also just from not
Open the detection technique to protein.Especially recently as the rapid development of genomics and proteomics so as to egg
The separation detection technique of white matter has higher requirement.
SDS- polyacrylamide gel electrophoresises (abbreviation SDS-page) are more normal in current Separation of Proteins identification experiment
Experimental technique.Polyacrylamide gel is network structure, has molecular sieving effect, SDS-PAGE is according only to protein subunit
The different can separating albumen matter of molecular weight, the electrophoretic mobility of protein subunit depend primarily on the size of molecular weight subunit
(electric charge factor can be ignored).
SDS is anionic detergent, and as denaturant and solubilizing agents, it can be broken intramolecular and intermolecular hydrogen bond,
Make molecule unfolding, destroy protein molecular two, tertiary structure.And strong reductant such as mercaptoethanol, dithiothreitol (DTT) can make half
Disulfide bonds between cystine residue.After adding reducing agent and SDS in sample and gel, molecule is depolymerizated into polypeptide chain,
Amino acid side chain and SDS after depolymerization are combined into albumen-SDS micellas, and the negative electrical charge of institute's band greatly exceed the original electricity of albumen
Lotus amount, thus eliminating the need the charge differences between different molecular and architectural difference.
Gel is made up of separation gel and concentration glue, and lower floor is separation gel, and gel aperture is smaller, there is molecular sieving effect, denaturation
Protein molecule institute is negatively charged basically identical, and mobility speed depends mainly on molecular weight.Upper strata is concentration glue, and there is accumulation to make
With gel strength is smaller, and aperture is larger, diluter sample is added on concentration glue, by the migration of large aperture gel
It is concentrated into a narrow zone.When sample liquid and concentration glue select TRIS/HCl buffer solutions, electrode solution selects TRIS/ glycine.
After electrophoresis starts, HCl is dissociated into chlorion, and glycine dissociates a small amount of glycine radical ion.Protein belt negative electrical charge, therefore
Moved together to positive pole, wherein chlorion is most fast, and glycine radical ion is most slow, and albumen is placed in the middle.Chlorion swimming when electrophoresis starts
Rate is maximum, more than albumen, therefore low conductance area is formed below, and electric-field intensity is inversely proportional with low conductance area, thus generation compared with
High electric-field intensity, albumen and glycine radical ion is moved rapidly, form a stable interface, albumen is gathered in mobile boundary
Near face, an intermediate layer is condensed into, substantially increases resolution ratio.
But in this method, after the completion of gel electrophoresis process, albumen is also needed with amino black, Coomassie brilliant blue, Silver stain
Agent etc. is dyed, and result is then observed after decolorization, and the ordinary stain time is 25min, and bleaching time is even more to be up to 12-
As long as 24h, experimentation is long and complicated, and coloring agent used also has certain pollution and harmfulness, limits SDS- polypropylene
Acrylamide gel electrophoresis further applying in protein analysis detection.
The content of the invention
For above-mentioned prior art, it is an object of the invention to provide a kind of new detection method of protein, this method makes
Compound is formed with specific fluorescer and protein binding, needs not move through traditional dyeing and decolorization process, can be with quick detection
Albumen, and detection time is short, testing cost is low, and reduce the pollution to environment.
To achieve the above object, the present invention adopts the following technical scheme that：
One kind detection method of protein, comprises the following steps：
(1) protein example is subjected to denaturation treatment, red sulphonyl is then added into the protein example after denaturation treatment
Solutions of chlorine, 20-120min is placed at 30-50 DEG C；
(2) SDS-PAGE is prepared, the protein example added after step (1) processing, carries out gel electrophoresis operation；
(3) after the completion of electrophoresis, protein adhesive is placed under uviol lamp or LED and observed, immediately arrives at testing result.
Preferably, in step (1), the method for protein example denaturation treatment is：By protein example at 90-120 DEG C
Handle 3-15min.
Preferably, in step (1), the solution of dansyl chloride is the supersaturated solution or red sulphur that dansyl Cl is prepared with water
The solution that acyl chlorides is prepared with organic solvent；The organic solvent is any in acetone, n-butanol, dichloromethane, acetonitrile or benzene
Kind；It is further preferred that the organic solvent is acetone or n-butanol.
It is more highly preferred to, the solution of dansyl chloride is the supersaturated solution that dansyl Cl is prepared with water.
Preferably, in the solution of dansyl chloride, the concentration of dansyl Cl is 0.0002-0.001g/ml；It is more highly preferred to
, in the solution of dansyl chloride, the concentration of dansyl Cl is 0.0003g/ml.
Preferably, in step (1), the ratio of solution of dansyl chloride and protein example addition is 5ml：(5-30)mg.
The present invention is had found during experiment, and fluorescence labeling processing, dansyl Cl are carried out to protein using dansyl Cl
The solvent species of solution, the concentration of solution of dansyl chloride, solution of dansyl chloride and the proportioning of protein, the temperature of processing and processing
Time etc. fluorescence labeling treatment effect can all be had an impact, be the key factor for influenceing final Protein Detection effect.
Wherein, for preparing solution of dansyl chloride used by solvent species, due to the huge number of solvent, including organic
Solvent and inorganic solvent, according to the deliquescent feature of dansyl Cl, the present invention is in optimized selection from organic solvent first, no
Congener organic solvent, on the one hand the solute effect of dansyl Cl is had differences；On the other hand, it is right after dissolving dansyl Cl
The treatment effect of protein fluorescence mark also has difference.Found through lot of experiments, it is organic molten with dichloromethane, acetonitrile or benzene etc.
Agent prepares solution as solvent, although there is preferable solute effect, can not have preferable fluorescence labeling treatment effect simultaneously, examines
It is unstable to survey effect, and bright band easily occurs；And using acetone and n-butanol as solvent, there is good solute effect, and compared with
Good fluorescence labeling treatment effect, but Detection results are still relatively less stable.Because dansyl Cl is water-insoluble, the present invention
The phase once forecloses water before the test, but the later stage is found surprisingly that, by dansyl Cl with water be configured to supersaturated solution (or
Be " suspension "), use it for protein fluorescence labeling processing, achieve unexpected effect, electrophoretic band is clear,
Detection results are stable, the situation of bright band no longer occur.Therefore, dansyl Cl is configured to supersaturated solution with water, then to albumen
Matter carries out the best results of fluorescence labeling processing；And using water as solvent safety, it is nontoxic, greatly reduce the contaminative of experiment
And harmfulness.
For the temperature of processing, temperature is too low to cause dansyl Cl from fully being marked to protein, temperature mistake
Gao Zehui destroys mark, and therefore, the selection for the treatment of temperature is very crucial, it is a discovery of the invention that when treatment temperature is 30-50 DEG C,
Treatment effect is preferable；Particularly, when treatment temperature is 37 DEG C, fluorescence labeling treatment effect is optimal.
For solution of dansyl chloride and the ratio of protein example addition, if solution of dansyl chloride addition is very few, can make
Dansyl Cl is obtained can not fully to mark protein；Solution of dansyl chloride addition is excessive, then can influence electrophoresis detection effect
Fruit.Through testing preferred discovery, when solution of dansyl chloride and the ratio of protein example addition are 5ml：It can be taken during (5-30) mg
Obtain optimal Protein Detection effect.
In addition, the opportunity for carrying out fluorescence labeling to protein using solution of dansyl chloride is also very crucial, the present invention is testing
During once selected respectively before albuminous degeneration and carry out fluorescence labeling again after gel electrophoresis, as a result Protein Detection result is paid no attention to
Think, observed under uviol lamp or LED, have no corresponding band and occur.
Preferably, in step (2), SDS-PAGE preparation includes：The preparation of separation gel and the preparation for concentrating glue.
Preferably, the compound method of separation gel is：By ddH2O 1.3ml, acrylamide-methylene diacrylamide (29:1)
1.65ml, 1.5M Tris-HCl (pH 8.8) 1.9ml, 10%SDS 0.05ml, 10%AP 0.05ml, TEMED0.05ml match somebody with somebody
Gel solution, is injected into the slit between long and short glass plate by material after mixing, in gel surface plus one layer of water to completely cut off air,
Make glue surface smooth, the water on upper strata is outwelled after colloid solidification.
Preferably, the compound method for concentrating glue is：By ddH2O 1.3ml, acrylamide-methylene diacrylamide (29:1)
0.3ml, 0.5M Tris-HCl (pH 6.8) 0.25ml, 10%SDS 0.06ml, 10%AP 0.06ml, TEMED0.04ml match somebody with somebody
Material, gel solution is injected above separation gel after mixing, add sample template comb, stand, to colloid solidification.
Beneficial effects of the present invention：
(1) present invention carries out fluorescence labeling processing using dansyl Cl to protein example, handles sample and SDS-PAGE
Preparation synchronously carry out, without the step of being dyed and be decolourized again after electrophoresis, compared with existing detection method, greatly shorten
Experiment duration, reduces experiment complexity.
(2) dansyl Cl used in the present invention is extremely micro, therefore compare existing detection method, its contaminative with
Harmfulness is much smaller, and reduces the cost of detection.
Brief description of the drawings
Fig. 1：The Protein Detection result of embodiment 1；In figure, swimming lane 1:55kDa, swimming lane 2:66kDa；Swimming lane 3：29kDa；Swimming
Road 4：Three kinds of mixed proteins.
Fig. 2：The Protein Detection result of comparative example 1；In figure, swimming lane 1:55kDa, swimming lane 2:66kDa；Swimming lane 3：29kDa；Swimming
Road 4：Three kinds of mixed proteins.
Fig. 3：The Protein Detection result of comparative example 2；In figure, swimming lane 1:55kDa, swimming lane 2:66kDa；Swimming lane 3：29kDa；Swimming
Road 4：Three kinds of mixed proteins.
Fig. 4：The Protein Detection result of comparative example 3；In figure, swimming lane 1:55kDa, swimming lane 2:66kDa；Swimming lane 3：29kDa；Swimming
Road 4：Three kinds of mixed proteins.
Fig. 5：The Protein Detection result of comparative example 4；In figure, swimming lane 1:55kDa, swimming lane 2:66kDa；Swimming lane 3：29kDa；Swimming
Road 4：Three kinds of mixed proteins.
Fig. 6：The Protein Detection result of comparative example 5；In figure, swimming lane 1:55kDa, swimming lane 2:66kDa；Swimming lane 3：29kDa；Swimming
Road 4：Three kinds of mixed proteins.
Fig. 7：The Protein Detection result of comparative example 6；In figure, swimming lane 1:55kDa, swimming lane 2:66kDa；Swimming lane 3：29kDa；Swimming
Road 4：Three kinds of mixed proteins.
Embodiment
It is noted that described further below is all exemplary, it is intended to provides further instruction to the application.It is unless another
Indicate, all technologies used herein and scientific terminology are with usual with the application person of an ordinary skill in the technical field
The identical meanings of understanding.
In order that the technical scheme of the application can clearly be understood by obtaining those skilled in the art, below with reference to tool
The embodiment of body describes the technical scheme of the application in detail.
Test material used is the conventional test material in this area in the embodiment of the present invention and comparative example, can be passed through
Commercial channel is commercially available.
Embodiment 1：
(1) processing of protein example：
It is respectively 55kDa, 66kDa, 29kDa standard protein with molecular weight, and the egg mix of these three standard proteins
It is used as protein example in vain.
First, denaturation treatment, i.e., 100 DEG C processing 5min are carried out to protein example；Then to the albumen after denaturation treatment
Quality sample solution add solution of dansyl chloride (be formulated by dansyl Cl and water, be supersaturated solution, concentration 0.0003g/
Ml), solution of dansyl chloride and the ratio of protein example addition are 5ml：10mg, 37 DEG C of placement 90min.
(2) SDS-PAGE is prepared：
1) preparation of separation gel：
The formula of 10% separation gel (5ml) is as follows：
ddH2O 1.3ml, acrylamide-methylene diacrylamide (29:1) 1.65ml, 1.5M Tris-HCl (pH 8.8)
1.9ml, 10%SDS 0.05ml, 10%AP 0.05ml, TEMED0.05ml.
Gel solution is injected into the slit between long and short glass plate after mixing, in gel surface plus one layer of water to completely cut off
Air, makes glue surface smooth, and the water on upper strata is outwelled after colloid solidification.
2) preparation of glue is concentrated：
The formula of 5% concentration glue (2ml) is as follows：
ddH2O 1.3ml, acrylamide-methylene diacrylamide (29:1) 0.3ml, 0.5M Tris-HCl (pH 6.8)
0.25ml, 10%SDS 0.05ml, 10%AP 0.06ml, TEMED0.04ml.
Gel solution is injected into the slit between long and short glass plate (above separation gel) after mixing, gently adds sample
Template is combed, and carefully avoids the appearance of bubble, about 30min, polymerization is completely.
(3) device, which is transferred in electrophoresis tank, adds electrophoresis liquid, extracts sample template comb, by the albumen sample after step (1) processing
Gel electrophoresis operation is carried out in product adding hole.
(4) after the completion of electrophoresis, protein adhesive is placed under uviol lamp (or LED) and observed, immediately arrives at experimental result.Knot
Fruit is as shown in Figure 1.
Comparative example 1：
(1) processing of protein example：
It is respectively 55kDa, 66kDa, 29kDa standard protein with molecular weight, and the egg mix of these three standard proteins
It is used as protein example in vain.Denaturation treatment, i.e., 100 DEG C processing 5min are carried out to protein example
(2) SDS-PAGE is prepared：
1) preparation of separation gel：
The formula of 10% separation gel (5ml) is as follows：
ddH2O 1.3ml, acrylamide-methylene diacrylamide (29:1) 1.65ml, 1.5M Tris-HCl (pH 8.8)
1.9ml, 10%SDS 0.05ml, 10%AP 0.05ml, TEMED0.05ml.
Gel solution is injected into the slit between long and short glass plate after mixing, in gel surface plus one layer of water to completely cut off
Air, makes glue surface smooth, and the water on upper strata is outwelled after colloid solidification.
2) preparation of glue is concentrated：
The formula of 5% concentration glue (2ml) is as follows：
ddH2O 1.3ml, acrylamide-methylene diacrylamide (29:1) 0.3ml, 0.5M Tris-HCl (pH 6.8)
0.25ml, 10%SDS 0.05ml, 10%AP 0.06ml, TEMED0.04ml.
Gel solution is injected into the slit between long and short glass plate (above separation gel) after mixing, gently adds sample
Template is combed, and carefully avoids the appearance of bubble, about 30min, polymerization is completely.
(3) device, which is transferred in electrophoresis tank, adds electrophoresis liquid, extracts sample template comb, by the albumen sample after step (1) processing
Product add aerial progress gel electrophoresis operation.
(4) after the completion of electrophoresis, protein adhesive is placed in coomassie brilliant blue R_250 dye liquor and dyes 25min, after taken off with destainer
Color is to it can be seen that result, the time is about 12-24h.As a result it is as shown in Figure 2.
Wherein, the composition of coomassie brilliant blue R_250 dye liquor is：
0.1% (W/V) coomassie brilliant blue R_250,25% (V/V) isopropanol, 10% (V/V) glacial acetic acid；
Compound method：
1) 1g coomassie brilliant blue R_250s are weighed, are placed in 1L beakers.
2) isopropanol for measuring 250ml is added in above-mentioned beaker, stirring and dissolving.
3) 100ml glacial acetic acid is added, is stirred.
4) 650ml deionized water is added, is stirred.
5) after removing particulate matter with filter paper, room temperature preservation.
Destainer formula constituent concentration：10% (V/V) acetic acid, 5% (V/V) ethanol
Compound method：
1) following solutions are measured, are placed in 1L beakers.
Acetic acid：100ml
Ethanol：50ml
dH2O：850ml
2) used after being sufficiently mixed.
It is molten using dansyl Cl it can be seen from Fig. 1 and Fig. 2 after to protein example denaturation treatment, before gel electrophoresis
Liquid carries out fluorescent staining (i.e. the embodiment of the present invention 1) to the protein example after denaturation, is contaminated relative to traditional Coomassie brilliant blue
Color (comparative example 1), without follow-up decolorization operations, substantially reduce the time of experiment；Moreover, come from last experimental result
See, compared with comparative example 1, the experimental result of embodiment 1 is more stable, clear.
Comparative example 2：
The solvent that solution of dansyl chloride is prepared in embodiment 1 is adjusted to " dichloromethane ", other are the same as embodiment 1；Electrophoresis
After the completion of, protein adhesive is placed under uviol lamp and observed, draws experimental result.As a result it is as shown in Figure 3.
Comparative example 3：
The concentration of solution of dansyl chloride in embodiment 1 is adjusted to " 0.0001g/ml ", other are the same as embodiment 1；Electrophoresis is complete
Cheng Hou, protein adhesive is placed under uviol lamp and observed, draws experimental result.As a result it is as shown in Figure 4.
Comparative example 4：
The concentration of solution of dansyl chloride in embodiment 1 is adjusted to " 0.002g/ml ", other are the same as embodiment 1；Electrophoresis is completed
Afterwards, protein adhesive is placed under uviol lamp and observed, draw experimental result.As a result it is as shown in Figure 5.
Comparative example 5：
The temperature placed after addition solution of dansyl chloride in embodiment 1 is adjusted to " 25 DEG C ", the time of placement is adjusted to
" 140min ", other are the same as embodiment 1；After the completion of electrophoresis, protein adhesive is placed under uviol lamp and observed, draws experimental result.As a result
As shown in Figure 6.
Comparative example 6：
The temperature placed after addition solution of dansyl chloride in embodiment 1 is adjusted to " 60 DEG C ", the time of placement is adjusted to
" 15min ", other are the same as embodiment 1；After the completion of electrophoresis, protein adhesive is placed under uviol lamp and observed, draws experimental result.As a result such as
Shown in Fig. 7.
Change the species for the solvent for preparing solution of dansyl chloride it can be seen from above-mentioned comparative example 2-6, change dansyl Cl
The conditions such as the temperature and time when concentration or change solution of dansyl chloride labelled protein of solution, all can strong influence albumen
The effect of fluorescence labeling, cause final Protein Detection result undesirable (Fig. 3-Fig. 7).As a result show：Not arbitrary red sulphur
Solution of acid chloride can be used in the fluorescence labeling of albumen, and dansyl Cl and water only are configured into supersaturated solution, and certain
Placed the regular hour under temperature conditionss, preferable fluorescence labeling effect and stable Protein Detection result could be obtained.
The preferred embodiment of the application is the foregoing is only, is not limited to the application, for the skill of this area
For art personnel, the application can have various modifications and variations.It is all within spirit herein and principle, made any repair
Change, equivalent substitution, improvement etc., should be included within the protection domain of the application.
Claims (10)
1. one kind detection method of protein, it is characterised in that comprise the following steps：
(1) protein example is subjected to denaturation treatment, it is molten that dansyl Cl is then added into the protein example after denaturation treatment
Liquid, 20-120min is placed at 30-50 DEG C；
(2) SDS-PAGE is prepared, the protein example added after step (1) processing, carries out gel electrophoresis operation；
(3) after the completion of electrophoresis, protein adhesive is placed under uviol lamp or LED and observed, immediately arrives at testing result.
2. according to the method for claim 1, it is characterised in that in step (1), the method for protein example denaturation treatment
For：Protein example is handled into 3-15min at 90-120 DEG C.
3. according to the method for claim 1, it is characterised in that in step (1), the solution of dansyl chloride is dansyl Cl
The supersaturated solution prepared with water；Or the solution that dansyl Cl is prepared with organic solvent.
4. according to the method for claim 3, it is characterised in that the organic solvent be acetone, n-butanol, dichloromethane,
Any of acetonitrile or benzene；Preferably, the organic solvent is acetone or n-butanol.
5. according to the method for claim 3, it is characterised in that the solution of dansyl chloride is that dansyl Cl is prepared with water
Supersaturated solution.
6. method according to claim 1 or 5, it is characterised in that in the solution of dansyl chloride, the concentration of dansyl Cl
For 0.0002-0.001g/ml, preferably 0.0003g/ml.
7. according to the method for claim 6, it is characterised in that in step (1), solution of dansyl chloride adds with protein example
The ratio for entering amount is 5ml：(5-30)mg.
8. according to the method for claim 1, it is characterised in that in step (2), SDS-PAGE preparation includes：Separation gel
Preparation and concentrate glue preparation.
9. according to the method for claim 8, it is characterised in that the compound method of separation gel is：By ddH2O 1.3ml, propylene
Acid amides-methylene diacrylamide (29:1) 1.65ml, 1.5M Tris-HCl (pH 8.8) 1.9ml, 10%SDS 0.05ml,
Gel solution, is injected into the slit between long and short glass plate by 10%AP 0.05ml, TEMED0.05ml dispensings after mixing,
Gel surface adds one layer of water to completely cut off air, makes glue surface smooth, and the water on upper strata is outwelled after colloid solidification.
10. according to the method for claim 8, it is characterised in that the compound method for concentrating glue is：By ddH2O 1.3ml, third
Acrylamide-methylene diacrylamide (29:1) 0.3ml, 0.5M Tris-HCl (pH 6.8) 0.25ml, 10%SDS 0.06ml,
10%AP 0.06ml, TEMED0.04ml dispensings, gel solution is injected above separation gel after mixing, adds sample template comb,
Stand, to colloid solidification.
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