CN102445487A - Electrophoretic analysis method for modified protein isolation and identification - Google Patents
Electrophoretic analysis method for modified protein isolation and identification Download PDFInfo
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- CN102445487A CN102445487A CN2011102994681A CN201110299468A CN102445487A CN 102445487 A CN102445487 A CN 102445487A CN 2011102994681 A CN2011102994681 A CN 2011102994681A CN 201110299468 A CN201110299468 A CN 201110299468A CN 102445487 A CN102445487 A CN 102445487A
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Abstract
The invention discloses an electrophoretic analysis method for modified protein isolation and identification. The method is characterized by being implemented by the following steps of: (1) preparing gel and a buffer solution, (a) a gel solution consists of 6-8M of urea, 4-8 percent of acrylamide, 20mM of EDTA (Ethylene Diamine Tetraacetic Acid), 90mM of Tris (Trihydroxymethyl aminomethane)-boric acid of which the pH is 8.4, AP (Ammonium Persulfate), distilled water and TEMED (Tetramethylethylenediamine), wherein in the acrylamide, the ratio of Acr to Bis is (30-40:1; (b) a loading buffer solution consists of 35-50 percent of cane sugar, 6-8M of urea, 20mM of EDTA and 90mM of Tris-boric acid of which the pH is 8.4; and (c) an electrode buffer solution consists of 25mE of EDTA and 112.5mM of Tris-boric acid of which the pH is 8.4; (2) conventionally cooling under the condition that the electrophoresis voltage is 100-180V and the length of a gel plate is 50-200 millimeters for 4-6 hours till the temperature is below 60 DEG C; and (3) dyeing the gel, decolorizing, imaging, judging a strip of modified proteins in reference to unmodified proteins, or quantifying the modifying degree of proteins by using QualityOne software. The method has simple steps and a remarkable ionophortic separation effect, and is convenient to operate; and the modifying degree of natural proteins can be analyzed.
Description
Technical field
The present invention relates to biological technical field, particularly a kind of method through electrophoretic separation modified protein and native protein.
Background technology
Protein post-translational modification is an importance in the Identification of Fusion Protein work.Posttranslational modification comprises formation of phosphorylation, glycosylation, chelated mineral, the sealing of N end, disulfide bond or the like.Prlmary structure of protein can be deduced from gene order, but the information of posttranslational modification almost can't only obtain from gene order.And well-knownly be that these modify for the function of the function of protein and even cell all very important, also often relevant with the generation of disease.Therefore, the protein of different modifying is separated and identifies, in protein group, particularly significant in the research of functional protein group.
The effective method of existing protein modified evaluation is a mass spectroscopy, and mass spectroscopy can be confirmed Phosphorylated Peptide very soon through the method that characteristic ion detects, and confirm phosphorylation site through tandem mass spectrum.Method through mass spectrum, proteolysis and glycosidase enzymolysis combine is sought glycopeptide, can identify glycosylation site.
The method of existing protein modified separation has chromatographic technique and electrophoretic techniques.The protein electrophoresis method that can separate modified protein comprises 2D-PAGE, IEF-PAGE.The 2D-PAGE electrophoresis is a two dimensional electrophoresis, mostly is meant first to for isoelectric focusing (isoelectric point information), and second to being SDS-PAGE electrophoresis (molecular weight information), electrically chargedly per sample separates with the difference of molecular weight.The IEF-PAGE electrophoresis is the one dimension electrophoresis, and different samples can be separated according to its isoelectric point difference.The IEF-PAGE electrophoresis can separate different modified protein of isoelectric point and native protein, but can not obtain the degree of modification information of this albumen.In addition, utilize the monoclonal antibody specific of preparation also can separate some modified protein; Use the modification part of fluorescence probe, isotope specific marker albumen, can the adorned degree of analyzing proteins.But these methods all have shortcoming separately, cost height, length consuming time, contaminated environment etc.
Under the prerequisite of the ornamental equivalent of clear and definite a kind of albumen and decorating site, desire is with the native protein molecule in the albumen with by the modified protein molecular separation, and definite albumen by degree of modification, in the above-mentioned several method, have only the 2D-PAGE electrophoresis to meet the demands.But 2D-PAGE is a two dimensional electrophoresis, and instrument and equipment is required height, complicated operation.The present invention has set up a kind of one dimension electrophoresis method, i.e. Urea-PAGE protein electrophoresis, realize in the albumen the native protein molecule with by the separating of modified protein molecule, and definite albumen by degree of modification.
Urea-polyacrylamide gel electrophoresis (Urea-PAGE) is the one dimension electrophoresis, and it is widely used in always separates single stranded DNA and RNA, adds 6-8 M Urea in the electrophoresis, can make the secondary structure sex change of DNA and RNA.The DNA or the RNA that separate 2-500bp according to the difference of molecular weight.Its step is simple, and is easy to operate, and cost is low.
Summary of the invention
The present invention has set up to native protein and the Urea-PAGE protein electrophoresis that is separated by modified protein in the albumen; Provide a kind of with the native protein molecule in the albumen with separated by the modified protein molecule, and according to confirmed by the shared ratio of modified protein molecule albumen by the electrophoresis new method of degree of modification.Urea-PAGE protein electrophoresis process comprises joins glue, preparation gel slab, last appearance, electrophoresis, dyeing, decolouring and several steps such as gel imaging and interpretation as a result.
Technical matters to be solved by this invention realizes through following steps.
1) configuration gel and damping fluid: a) gel solution: 6-8 M urea; The acrylic amide of 4-8%; Acrylic amide (Acr): N in the acrylic amide wherein; The two propylene polyamide (Bis) of N-methene=30-40:1,20mM ethylenediamine tetraacetic acid (EDTA), the trishydroxymethylaminomethane of the pH8.4 of 90mM (Tris)-boric acid and ammonium persulfate (AP), distilled water, tetramethylethylenediamine (TEMED); B) trishydroxymethylaminomethane (Tris)-boric acid of the pH8.4 of the sucrose of sample-loading buffer: 35-50%, 6-8M urea, 20mM ethylenediamine tetraacetic acid (EDTA) and 90mM; C) electrode buffer: trishydroxymethylaminomethane (Tris)-boric acid of the pH8.4 of 25mM ethylenediamine tetraacetic acid (EDTA), 112.5mM.
2) electrophoresis: deposition condition is voltage 100-180 V, and electrophoresis time 4-6 hour, gel slab length was 50-200mm.Adopt the conventional method cooling during electrophoresis, electrophoresis temperature is controlled at below 60 ℃.
3) judgement of Separation of Proteins and modification: gel is dyed behind the electrophoresis, the imaging of decolouring back, and analyzing proteins is modified situation.If albumen has only a band in the SDS-PAGE protein electrophoresis, and go out two bands through this protein electrophoresis is separable, promptly decidable albumen has part to be modified.Contrast unmodified protein, can judge the band of modified protein, can also quantitatively go out the adorned degree of albumen through Quality One software.
The present invention uses conventional one dimension protein electrophoresis system, in gel, add Urea and make albuminous degeneration, and factors such as time through control albumen swimming speed, electrophoresis and voltage, with the native protein molecule in the albumen with separated by the modified protein molecule.Note in the electrophoresis process that must add Urea in gel and the sample-loading buffer makes albuminous degeneration, whole electrophoresis all can not add SDS influences the protein band quantity of electric charge, and this is the key of Urea-PAGE protein electrophoresis.
The Urea-PAGE protein electrophoresis that the present invention sets up, have following advantage: step is simple, and is easy to operate, only needs a kind of separation gel of preparation just can destination protein be separated; Required reagent all is conventional reagent, and uses conventional SDS-PAGE electrophoresis apparatus just can operate; The electrophoretic separation effect is obvious, and after electrophoresis finished, native protein molecule and modified protein molecule were in different swimming lane positions because of swimming speed is different; Through this protein electrophoresis, can also analyze the adorned degree of native protein.
Description of drawings
Fig. 1 modifies for the present invention's 7 M Urea-6% PAGE electrophoretic analysis ovotransferrins.Swimming lane 1:5 μ g Apo-OVT, swimming lane 2:5 μ gHolo-OVT.
Fig. 2 modifies for the present invention 6.5 M Urea-5% PAGE electrophoretic analysis ovotransferrins.Swimming lane 1-5: iron-free OVT and Fe
3+Ratio be followed successively by 1:0,2:1,1:1,1:2,1:5, every hole applied sample amount is 5 μ g.
Embodiment
The present invention further specifies through following examples, but the present invention is not limited.
Embodiment 1.
The separation of ferric ion chelating ovotransferrins (Holo-OVT) and iron-free ovotransferrins (Apo-OVT).
Adopt Urea-PAGE protein electrophoresis separation of iron ion modification ovotransferrins (Holo-OVT) and iron-free ovotransferrins (Apo-OVT); Their molecular weight is about 76KDa; Ovotransferrins is after ferric ion is modified; Since the ferric ion positively charged, a part of negative charge in the albumen that neutralized, and ovotransferrins (Holo-OVT) is electronegative to be lacked than iron-free ovotransferrins (Apo-OVT) so ferric ion is modified.
The practical implementation process comprises the steps.
1, configuration gel and damping fluid.
Contain 7M Urea in the gel solution, and 6% acrylic amide (Acr:Bis=37.5:1), 20mM EDTA, 90mM Tris-boric acid (pH8.4) and AP, ddH2O, TEMED.
Protein solution contains 50% sucrose, 7M Urea, 20mM EDTA and 90mM Tris-boric acid (pH8.4) with after the sample-loading buffer equal-volume mixes in the sample.
Contain 25mM EDTA, 112.5mM Tris-boric acid (pH8.4) in the electrode buffer.
2, go up appearance and electrophoresis: adopt the Bio-Rad gel electrophoresis system, gel slab length is 74mm.Sample and equal-volume 2 * sample buffer mix the centrifugal 1min in back, in addings in kind hole.Deposition condition is voltage 150 V, electrophoresis time 4 hours.Be equipped with recirculated cooling water during electrophoresis, electrophoresis temperature is controlled at below 60 ℃.
3, the separation of protein and modification are judged.
After electrophoresis finishes, gel is placed in the double dish, adds dyeing liquor, shaking table vibration dyeing 30min, clear with the destainer band that decolours to the protein district then.Place gel imaging system to form images gel at last, according to electrophoretogram analytical electrophoresis result.
The electrophoresis picture of swimming lane 1-2 is shown in accompanying drawing 1.By finding out that Apo-OVT is different with Holo-OVT swimming speed among the figure, Holo-OVT is faster than the swimming speed of Apo-OVT, and two bands appear in this swimming lane, at a distance of about 1-2cm.Hence one can see that, and iron-free OVT band is natural OVT, Fe
2The OVT that-OVT band is modified by ferric ion, Apo-OVT does not have the chelated iron ion, but holo-OVT is not two ferric ions of chelating of 100%.Can know that by Quality One software analysis Holo-OVT has 90.8% to be modified by ferric ion; Apo-OVT is the protein molecular of no ferric ion.
Embodiment 2.
The quantitative test of ovotransferrins (OVT) degree of modification.
The ovotransferrins of ferric ion that contains variable concentrations is as by the protein sample analyzed, through the Urea-PAGE protein electrophoresis with iron-free ovotransferrins (OVT) and ferric ion modification ovotransferrins (Fe
2-OVT) separate.Through Quality One software analysis Fe
2-OVT accounts for the ratio of whole ovotransferrins, i.e. the degree of modification of quantitative test ovotransferrins.
The practical implementation process comprises the steps.
1, configuration gel and damping fluid.
Contain 6.5M Urea in the gel solution, and 5% acrylic amide (Acr:Bis=32.5:1), 20mM EDTA, 90mM Tris-boric acid (pH8.4) and AP, ddH
2O, TEMED.
Protein solution contains 40% sucrose, 6.5M Urea, 20mM EDTA and 90mM Tris-boric acid (pH8.4) with after the sample-loading buffer equal-volume mixes in the sample.
Contain 25mM EDTA, 112.5mM Tris-boric acid (pH8.4) in the electrode buffer.
2, go up appearance and electrophoresis: adopt the Bio-Rad gel electrophoresis system, gel slab length is 74mm.Sample and equal-volume 2 * sample buffer mix the centrifugal 1min in back, in addings in kind hole.Deposition condition is voltage 125 V, electrophoresis time 5 hours.Be equipped with recirculated cooling water during electrophoresis, electrophoresis temperature is controlled at below 60 ℃.
3, the separation of protein and modification are judged.
After electrophoresis finishes, gel is placed in the double dish, adds dyeing liquor, shaking table vibration dyeing 30min, clear with the destainer band that decolours to the protein district then.Place gel imaging system to form images gel at last, according to electrophoretogram analytical electrophoresis result.
The electrophoresis picture of swimming lane 1-5 is shown in accompanying drawing 2.That separates between each band of albumen is fine, can significantly find out, along with Fe
3+Concentration increase, iron-free OVT content reduces Fe gradually
2-OVT content increases gradually.Promptly along with Fe
3+Increase, the OVT that ferric ion is modified increases gradually, still, can be known by two protein bands in the swimming lane 5, even add the Fe of 5 times of protein contents
3+With the OVT effect, OVT is 100% modified by ferric ion not.Can know that by Quality One software analysis from swimming lane 1-5, the OVT that is modified by ferric ion accounts for 0%, 29.8%, 82.4%, 92% and 95.2% of whole OVT respectively.
Claims (1)
1. one kind is used for by the electrophoresis analytical method of modified protein isolation identification, it is characterized in that realizing through following steps:
1) configuration gel and damping fluid: a) gel solution: 6-8 M urea; The acrylic amide of 4-8%; Acrylic amide in the acrylic amide: N wherein; The two propylene polyamide=30-40:1 of N-methene, 20mM ethylenediamine tetraacetic acid, the trishydroxymethylaminomethane-boric acid of the pH=8.4 of 90mM and ammonium persulfate, distilled water, tetramethylethylenediamine; B) trishydroxymethylaminomethane-boric acid of the pH8.4 of the sucrose of sample-loading buffer: 35-50%, 6-8M urea, 20mM ethylenediamine tetraacetic acid and 90mM; C) electrode buffer: trishydroxymethylaminomethane-boric acid of the pH8.4 of 25mM ethylenediamine tetraacetic acid, 112.5mM;
2) electrophoresis: deposition condition is voltage 100-180 V, and electrophoresis time 4-6 hour, gel slab length was 50-200mm; Adopt the conventional method cooling during electrophoresis, electrophoresis temperature is controlled at below 60 ℃;
3) judgement of Separation of Proteins and modification: gel is dyed behind the electrophoresis, and the imaging of decolouring back contrasts unmodified protein, judges the band of modified protein, or quantitatively goes out the adorned degree of albumen through Quality One software.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104661731A (en) * | 2012-08-07 | 2015-05-27 | 生物辐射实验室股份有限公司 | Modified electrode buffers for stain-free protein detection in electrophoresis |
| CN104698057A (en) * | 2015-03-25 | 2015-06-10 | 河南科技大学 | Antimicrobial experimenting method capable of determining antimicrobial protein fragment molecular weight |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1327537A (en) * | 1999-12-02 | 2001-12-19 | 海茂株式会社 | Polyacrylamide precast gels for electrophoresis, process for producing the same and electrophoresis method by using the gels |
| US20030121784A1 (en) * | 1994-03-31 | 2003-07-03 | Invitrogen Corporation | System for pH-neutral stable electrophoresis gel |
| CN101768207A (en) * | 2009-01-05 | 2010-07-07 | 中国医学科学院基础医学研究所 | Protein complex or compound three-dimensional gel electrophoresis separating method |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030121784A1 (en) * | 1994-03-31 | 2003-07-03 | Invitrogen Corporation | System for pH-neutral stable electrophoresis gel |
| CN1327537A (en) * | 1999-12-02 | 2001-12-19 | 海茂株式会社 | Polyacrylamide precast gels for electrophoresis, process for producing the same and electrophoresis method by using the gels |
| CN101768207A (en) * | 2009-01-05 | 2010-07-07 | 中国医学科学院基础医学研究所 | Protein complex or compound three-dimensional gel electrophoresis separating method |
Non-Patent Citations (1)
| Title |
|---|
| FUMIYASU FUKUMORI 等: "《Purification and Characterization of 2,4-Dichlorophenoxyacetate/a-Ketoglutarate Dioxygenase》", 《THE JOURNAL OF BIOLOGICAL CHEMISTRY》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104661731A (en) * | 2012-08-07 | 2015-05-27 | 生物辐射实验室股份有限公司 | Modified electrode buffers for stain-free protein detection in electrophoresis |
| CN104661731B (en) * | 2012-08-07 | 2017-03-01 | 生物辐射实验室股份有限公司 | For exempting from the improved electrode buffer of dsred protein detection in electrophoresis |
| CN104698057A (en) * | 2015-03-25 | 2015-06-10 | 河南科技大学 | Antimicrobial experimenting method capable of determining antimicrobial protein fragment molecular weight |
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