CN1159579C - Macromolecular weight protein reference material for active electrophoresis - Google Patents
Macromolecular weight protein reference material for active electrophoresis Download PDFInfo
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- CN1159579C CN1159579C CNB01136534XA CN01136534A CN1159579C CN 1159579 C CN1159579 C CN 1159579C CN B01136534X A CNB01136534X A CN B01136534XA CN 01136534 A CN01136534 A CN 01136534A CN 1159579 C CN1159579 C CN 1159579C
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- reference material
- damping fluid
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- tubulin
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Abstract
The present invention provides a high molecular weight protein standard material for active electrophoresis, which is the aqueous solution of microtubulin. The invention provides the purpose of the microtubulin taken as the high molecular weight protein standard material.
Description
Invention field
The present invention relates to a kind of macromolecular weight protein reference material that is used for active electrophoresis.The invention still further relates to the purposes of tubulin as macromolecular weight protein reference material.
Background of invention
Natural, polycomponent macromolecular weight protein reference material is the biological reagent commonly used of identification of organism sample reactive protein.High molecular reference material commonly used at present contains component more than 5 kinds, and these components are all extracted respectively from various biomaterials, purifying, is mixed with by a certain percentage then to form.Different method of purifying protein differences, agents useful for same, instrument difference, so product price is very expensive.The present invention can go out a kind of highly purified tubulin by fast purifying from animal brain, utilize the diversity of its oligomer, the disposable high-purity that contains component more than 6 kinds at least, the macromolecular weight protein reference material of high stability prepared.Productive rate is the 16-20mg/100g brain tissue; Purity reaches more than 95%; Molecular weight ranges is 55kDa-330kD or wideer; The active principle molecular weight is followed successively by 55kDa, 110kDa, 165kDa, 220kDa, 275kDa, 330kDa ...This simple technical method is easy to grasp; Used instrument, reagent type are few, are easy to investment; Product property is stable, is easy to preserve; Electrophoretic band is clear, and qualification result is accurate.
Summary of the invention
An object of the present invention is to provide a kind of macromolecular weight protein reference material that is used for active electrophoresis.
Another object of the present invention provides a kind of purposes of tubulin.
Tubulin is a kind of water-soluble globular protein that keeps very much in the biological cell, and molecular weight is 55kDa.People mainly concentrate on the research of tubulin in its fundamental research of structure, nature and function.Past thinks that always tubulin generally exists with dimeric forms in external buffer solution; 25 ℃ of degree to 37 ℃ temperature ranges, when guanopterin nucleoside triphosphate (GTP) is provided (or other conditions), the continuous polymerization of tubulin dimer meeting forms long microtubule at last.
We find under study for action: tubulin not only has dimeric forms to exist in aqueous solution (as: the PEM damping fluid that the purifying tubulin is used, or the sample buffer used of conventional electrophoresis), also has monomer and various forms of oligomer to exist; And between these multi-form tubulin polymerization bodies, the stepped increase of molecular weight, the adjacent ribbons difference is 55kDa; These existence forms of tubulin are insensitive to the variation of temperature and pH, when pH is 7-10, no matter carry out electrophoresis under common refrigerator (4-8 °) condition, still carry out electrophoresis at room temperature condition, can both obtain good result.Our tubulin product that studies have shown that purifying from fresh brain tissue, very suitable molecular weight standard thing as non-denatured protein matter electrophoresis.
With method purifying tubulin from fresh brain tissue of polymerization-depolymerization, can be with reference to the classic method (Meth.Enzymol, 1982,85, Part B, 376) of reports such as Williams.Used technology is a routine techniques, and method is very ripe, by expert, scholar extensively quote both at home and abroad.
Thereby, the invention provides a kind of macromolecular weight protein reference material that is used for active electrophoresis, it is the aqueous solution of tubulin.Wherein, described pH value of aqueous solution is preferably 7-10.Can be piperazine-N ', N-two 2-ethanesulfonic acid (PEM) damping fluid, phosphate buffer, protein electrophorese damping fluid or Tris-HCl damping fluids.They these damping fluids are had no particular limits, as long as can be adjusted to solution needed pH value.
The present invention also provides the purposes of tubulin as macromolecular weight protein reference material.Wherein, described tubulin is present in the aqueous solution, for example, can be the PEM damping fluid, and phosphate buffer is in protein electrophorese damping fluid or the Tris-HCl damping fluid.
Than other molecular weight standard thing in the market, our product advantage is: only with a kind of material, a cover technology, once experiment, just can obtain to contain the above product of 6 kinds of different molecular weight components.And other molecular weight standard thing must be with multiple material, many covers technology, repeatedly experiment could obtain.
Brief Description Of Drawings
Fig. 1 shows the purity evaluation of pig brain tubulin.Wherein, road 1, molecular weight standard thing; Road 2, thick product; Road 3, finished product; Road 4, Western blotting is identified.
Fig. 2 shows the molecular weight distribution (freshly prepd product) of this product under the condition of different pH.Wherein, A. is with the product B of PIPES damping fluid preparation. contain the product of 25% glycerine.The macromolecular weight protein reference material 1-5 of M:AmershamPharmacia Biotech company: sample pH value is followed successively by 5,7 on the product, and 9,10,12.
Fig. 3 shows the electrophoresis behavior of-20 ℃ of products of preserving under condition of different pH.Wherein, A. is with the product B of PIPES damping fluid preparation. contain the product of 25% glycerine.The macromolecular weight protein reference material 1-5 of M:AmershamPharmacia Biotech company: the electrophoresis behavior of this product under different pH values (5,7,9,10,12).
Fig. 4 shows the electrophoresis behavior (20 ℃ of products of preserving) of this product under the different temperatures deposition condition.Wherein, electrophoresis under ℃ following electrophoresis B. room temperature A.4.The macromolecular weight protein reference material 1-5:pH of M:Amersham PharmaciaBiotech company value is respectively 5,7,9,10,12
Embodiment
One, the required reagent of preparation of product
1.PEM damping fluid:
0.1mol/L?PIPES，PH6.9
2mmol/L?EGTA
1mmol/L?MgCl
2
1mmol/L?DTT
1mmol/L?PMSF
2.PEM-4M damping fluid:
Contain glycerine 4mmol/L in the PEM damping fluid
3.PEM-8M damping fluid:
Contain glycerine 8mmol/L in the PEM damping fluid
4.PEMG damping fluid:
Damping fluid contains GTP 0.1mmol/L among the PEM
5.0.1M PEM damping fluid:
0.1mol/L?PIPES，PH6.9
1mmol/L?EGTA
05mmol/L?MgCl
2
6,1M PMGD damping fluid:
1mol/L?PIPES，PH6.9
1.5mmol/L?MgCl
2
2mmol/L?GTP
20％DMSO
Two, required instrument
Balance, homogenizer, refrigerator, water-bath, high speed freezing centrifuge, ultracentrifuge.
Three, technological process
4 ℃ of homogenate → 4 ℃ high speed centrifugation → 4 ℃ of ultracentrifugation → 37 ℃ water-bath → 30 ℃ of ultracentrifugation → ice bath → 4 ℃ ultracentrifugation → 37 ℃ of water-bath → 30 ℃ ultracentrifugation → ice bath → 4 ℃ of ultracentrifugation → 37 ℃ water-bath → 30 ℃ of ultracentrifugation → ice bath → 4 ℃ ultracentrifugations
Four, operation steps
(1) preliminary purification of natural polymer amount reference material
1. get three fresh pig brains (about 300 grams), remove surface film, blood vessel and fat, the redistilled water with precooling washes then.
2. the PEM-4M damping fluid that adds 4 ℃ of precoolings of 240ml in tissue refiner shreds the pig brain and is placed on wherein homogenate.Homogenate was placed 30 minutes at 4 ℃.
3. homogenate is descended 10, centrifugal 30 minutes of 000g at 4 ℃.Get supernatant, 4 ℃ are descended 100, centrifugal 1 hour of 000g.
4. measure supernatant volume V
1, add ATP to 2.5mmol/L, GTP to 1mmol/L, polymerization was carried out in 37 ℃ of water-baths in 1 hour.
5. polymeric solution descends 100, centrifugal 1 hour of 000g at 30 ℃.
With resolution of precipitate at 1/4 V
1In the ice-cold PEMG damping fluid, depolymerization 1 hour (useable glass tissue grinder promotes depolymerization) in the frozen water.
7. depolymerization solution is descended 100, centrifugal 1 hour of 000g at 4 ℃.Get supernatant, add equal-volume PEM-8M damping fluid, and add GTP to 1mmol/L, after polymerization was carried out in 37 ℃ of water-baths in 1 hour.
8. after polymerization solution is descended 100, centrifugal 1 hour of 000g at 30 ℃.Abandon supernatant, stay precipitation.
9. precipitation is dissolved in the 0.1M PEM damping fluid of 4 ℃ of precoolings, depolymerization is 1 hour in the frozen water.
10. depolymerization solution is descended 100, centrifugal 20 minutes of 000g at 4 ℃.Supernatant is through two polymerizations-thick product of depolymerization round-robin.
(2) natural polymer amount protein standard thing is further purified
1. add equal-volume PMGD damping fluid in thick product, polymerization was carried out in 37 ℃ of water-baths in 30 minutes.
2. polymeric solution is descended 50, centrifugal 30 minutes of 000g at 30 ℃.Abandon supernatant, measure precipitation volume (V
2)
3. in precipitation, add 4V
20.1M PEM damping fluid, depolymerization is 1 hour in the frozen water water-bath.
4. depolymerization solution is descended 50 at 4 ℃, centrifugal 20 minutes of 000g, supernatant are the high-purity finished product.
(3) determination of protein concentration
According to a conventional method finished product is carried out determination of protein concentration, calculating must be measured.General 100 Borneo camphors are organized and are obtained the pure product of 16mg at least.
Five, product is preserved and operation instruction
(1) product is preserved: the product that purifying obtains is an aqueous solution, according to the protein concentration result who records above, the product dilution (is noted adding glycerine for 2mg/ml in the dilution; making the glycerine final concentration is 25%; because glycerine has protective effect), packing then ,-20 ℃ of preservations, for sale.
(2) description of product: this macromolecular weight protein reference material contains a kind of albumen, i.e. tubulin is for water-soluble.Separate demonstration through the polyacrylamide gel active electrophoresis, the actual active principle of this product is more than 6 kinds, and molecular weight is followed successively by 55kDa, 110kDa, 165kDa, 220kDa, 275kDa, 330kDa etc. from low to high.Adjacent ribbons differs 55kDa.
(3) using method: during use, add 5 times common non-sex change electrophoresis sample loading buffer (0.31MTris-HCl, pH 6.8; Glycerine 50%, bromjophenol blue 0.05%).Make macromolecular weight protein reference material concentration reach 1.5mg/ml.Applied sample amount 10 μ g-20 μ g/ roads are looked running gel and are varied in size.
Six, product is identified
(1) product purity and molecular weight identification
Utilize SDS-polyacrylamide gel sex change electrophoresis to identify purity.Resolving gel concentration 7.5%.Find out in the thick product micro-high molecular foreign protein is arranged still from Fig. 1 (road 2).But through being further purified, finished product is electrophoresis one band (Fig. 1, road 3), and molecular weight subunit is 55kDa.
(2) the molecular weight product active electrophoresis is analyzed
The active electrophoresis separation gel is 7.5%.
Find out from Fig. 2 (road 2, road 3), when pH is 7-9, at least 6 bands clearly appear from bottom to top in product, distribute (road M) evenly than Sigma company product banding pattern, and molecular weight ranges is moderate, is respectively 55kDa, 110kDa, 165kDa, 220kDa, 275kDa, 330kDa.Can also see that molecular weight is 385kDa, the band of 440kDa in addition.
(3) product pH stability analysis
Be respectively 5.0,7.0 with the pH value, 9.0,10.0,12 0.1M PEM damping fluid dissolved product precipitation, centrifugal after the depolymerization in the frozen water, the gained supernatant is the sample under the condition of different pH.Under 4 ℃ it is carried out active electrophoresis.From Fig. 2,3,4 road 1-5 finds out that product is insensitive to soda acid, shows good pH stability.Therefore, can guarantee under the situation of conventional electrophoresis pH reference material variation such as do not take place that component is dissociated.
(4) product is to the analysis of refrigerated storage stability
General reactive protein product can long-term frozen be preserved in 25% glycerine.(Fig. 3 is not A) with (Fig. 3, B), the protein molecular weight collection of illustrative plates distributes constant this product under the situation that glycerine is arranged there being glycerine.Right-20 ℃ of frozen good stable that show of product prove can-20 ℃ frozen.
(5) product is to the stability analysis of electrophoresis temperature
The above results is verified, and product during electrophoresis, shows good stability under 4 ℃ of conditions.During electrophoresis, find out that at room temperature the molecular weight distribution of product does not change, and proves that product is to the electrophoresis temperature wide adaptability from Fig. 4 B.
Seven, attached other similar products reference price
(1) Sigma (1999) MW-ND-500KIT FOR Molecular Weights (14,000-500,000), the 5mg packing, price is 80.30 dollars.Contain 7 kinds of components, molecular weight is respectively 14kDa-29kDa-45kDa-66kDa-132kDa-272kDa-545kDa.
(2) Amersham Pharmacia Biotech (calendar year 2001) HMW Native Marker kit, the 2.5mg packing, price is 115 dollars.Contain 5 kinds of components, molecular weight is respectively 66kDa-140kDa-232kDa-440kDa-669kDa.
Claims (6)
1. macromolecular weight protein reference material that is used for active electrophoresis, it is the aqueous solution of tubulin.
2. according to the described macromolecular weight protein reference material of claim 1, wherein, described pH value of aqueous solution is 7-10.
3. according to the described macromolecular weight protein reference material of claim 1, wherein, described aqueous solution is piperazine-N ', N-two 2-ethanesulfonic acid damping fluid, phosphate buffer, protein electrophorese damping fluid or Tris-HCl damping fluids.
4. tubulin is as the purposes of macromolecular weight protein reference material.
5. according to the described purposes of claim 4, wherein, described tubulin is present in the aqueous solution.
6. according to the described purposes of claim 4, wherein, described tubulin is present in the two 2-ethanesulfonic acid damping fluids of piperazine-N ', N-, and phosphate buffer is in protein electrophorese damping fluid or the Tris-HCl damping fluid.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB01136534XA CN1159579C (en) | 2001-10-15 | 2001-10-15 | Macromolecular weight protein reference material for active electrophoresis |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB01136534XA CN1159579C (en) | 2001-10-15 | 2001-10-15 | Macromolecular weight protein reference material for active electrophoresis |
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| CN1412558A CN1412558A (en) | 2003-04-23 |
| CN1159579C true CN1159579C (en) | 2004-07-28 |
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| CNB01136534XA Expired - Fee Related CN1159579C (en) | 2001-10-15 | 2001-10-15 | Macromolecular weight protein reference material for active electrophoresis |
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| CN101339159B (en) * | 2008-08-28 | 2012-05-02 | 内蒙古蒙牛乳业（集团）股份有限公司 | Milk alpha-casein content checking method |
| CN101339158B (en) * | 2008-08-28 | 2012-05-02 | 内蒙古蒙牛乳业（集团）股份有限公司 | Milk beta-casein content checking method |
| CN101556287B (en) * | 2009-02-26 | 2013-05-15 | 杭州松华生物科技有限公司 | Novel protein molecular weight standard and preparation method thereof |
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