CN100494962C - Wheat high-low molecular weight glutenin subunit separating analytical method - Google Patents
Wheat high-low molecular weight glutenin subunit separating analytical method Download PDFInfo
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- CN100494962C CN100494962C CNB2005100482147A CN200510048214A CN100494962C CN 100494962 C CN100494962 C CN 100494962C CN B2005100482147 A CNB2005100482147 A CN B2005100482147A CN 200510048214 A CN200510048214 A CN 200510048214A CN 100494962 C CN100494962 C CN 100494962C
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- 108010050792 glutenin Proteins 0.000 title claims abstract description 39
- 241000209140 Triticum Species 0.000 title claims description 39
- 235000021307 Triticum Nutrition 0.000 title claims description 39
- 238000004458 analytical method Methods 0.000 title description 3
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims abstract description 33
- 238000000034 method Methods 0.000 claims abstract description 24
- 102000006395 Globulins Human genes 0.000 claims abstract description 11
- 108010044091 Globulins Proteins 0.000 claims abstract description 11
- 108010088751 Albumins Proteins 0.000 claims abstract description 10
- 102000009027 Albumins Human genes 0.000 claims abstract description 10
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 10
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 10
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 10
- DNJIEGIFACGWOD-UHFFFAOYSA-N ethyl mercaptane Natural products CCS DNJIEGIFACGWOD-UHFFFAOYSA-N 0.000 claims abstract description 8
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 claims abstract description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 6
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 claims description 13
- RCEAADKTGXTDOA-UHFFFAOYSA-N OS(O)(=O)=O.CCCCCCCCCCCC[Na] Chemical compound OS(O)(=O)=O.CCCCCCCCCCCC[Na] RCEAADKTGXTDOA-UHFFFAOYSA-N 0.000 claims description 11
- 235000018102 proteins Nutrition 0.000 claims description 9
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 8
- 239000006228 supernatant Substances 0.000 claims description 8
- UXFQFBNBSPQBJW-UHFFFAOYSA-N 2-amino-2-methylpropane-1,3-diol Chemical compound OCC(N)(C)CO UXFQFBNBSPQBJW-UHFFFAOYSA-N 0.000 claims description 7
- 150000001875 compounds Chemical class 0.000 claims description 7
- 108010068370 Glutens Proteins 0.000 claims description 6
- 235000021312 gluten Nutrition 0.000 claims description 6
- UDSAIICHUKSCKT-UHFFFAOYSA-N bromophenol blue Chemical compound C1=C(Br)C(O)=C(Br)C=C1C1(C=2C=C(Br)C(O)=C(Br)C=2)C2=CC=CC=C2S(=O)(=O)O1 UDSAIICHUKSCKT-UHFFFAOYSA-N 0.000 claims description 4
- 235000011187 glycerol Nutrition 0.000 claims description 4
- 240000000581 Triticum monococcum Species 0.000 claims description 3
- 238000001816 cooling Methods 0.000 claims description 3
- 238000002156 mixing Methods 0.000 claims description 3
- 239000012488 sample solution Substances 0.000 claims description 3
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 claims description 3
- 239000013049 sediment Substances 0.000 claims description 2
- FVAUCKIRQBBSSJ-UHFFFAOYSA-M sodium iodide Chemical compound [Na+].[I-] FVAUCKIRQBBSSJ-UHFFFAOYSA-M 0.000 claims description 2
- 238000001962 electrophoresis Methods 0.000 abstract description 9
- SNRUBQQJIBEYMU-UHFFFAOYSA-N Dodecane Natural products CCCCCCCCCCCC SNRUBQQJIBEYMU-UHFFFAOYSA-N 0.000 abstract 1
- 229920002401 polyacrylamide Polymers 0.000 abstract 1
- 229910052708 sodium Inorganic materials 0.000 abstract 1
- 239000011734 sodium Substances 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 16
- 238000002360 preparation method Methods 0.000 description 7
- 238000000926 separation method Methods 0.000 description 5
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 4
- 229910052938 sodium sulfate Inorganic materials 0.000 description 4
- 235000011152 sodium sulphate Nutrition 0.000 description 4
- 239000007983 Tris buffer Substances 0.000 description 3
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 3
- 238000009395 breeding Methods 0.000 description 2
- 230000001488 breeding effect Effects 0.000 description 2
- 230000004069 differentiation Effects 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 230000002068 genetic effect Effects 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 108010061711 Gliadin Proteins 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000000840 electrochemical analysis Methods 0.000 description 1
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 230000009182 swimming Effects 0.000 description 1
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- Peptides Or Proteins (AREA)
Abstract
The invention supplies a method to separating analyze the high and low molecular weight glutenin subunit that adds isopropyl alcohol to remove the alcohol soluble protein, adds NaI to remove globulin, adds water to remove albumin, adds DDT, VP and mercaptoethanol to break the combination of disulfide bond. The invention lays a good foundation for testing electrophoresis by using sodium dodecyl sulfate-polyacrylamide gel.
Description
Technical field
The present invention relates to a kind of method of measuring high and low molecular weight glutenin subunit in the wheat, belong to electrochemical analysis and separation method technical field.
Background technology
Wheat gluten mainly contains glutenin, alcohol soluble protein, albumin and globulin four parts and forms.Wherein the high and low molecular weight glutenin subunit of wheat has decisive role to wheat quality, it not only influences the elasticity and the ductility of formation time, stabilization time and dough of precipitation number, wet gluten content, the dough of wheat, and the baking properties of wheat is also had decisive action.But in the electrophoresis analytical method of high and low molecular weight glutenin subunit to wheat, because the existence of albumin and globulin has influenced its electrophoresis, and because low molecular weight glutenin is close with the wheat gliadin molecular weight, therefore particularly nearly strip-type and main concentrated distribution surplus in the of 20 of low molecular weight glutenin subunit band be difficult to differentiation in the swimming collection of illustrative plates.The solution formula that is usually used in separating the high low molecular weight glutenin subunit of wheat and other albumen at present is to contain 0.625M2-amino-2 methylol-1, ammediol (Tris) (ph=6.8), the extract of 2% lauryl sodium sulfate (SDS), 0.3% bromophenol blue, 5% mercaptoethanol and 10% glycerine.Though this solution can extract the high and low molecular weight glutenin of wheat, also can remove albumin, but can not remove alcohol soluble protein and globulin, the combination of only breaking disulfide bond between each subunit in this solution in addition with mercaptoethanol, effect is insufficient, therefore in electrophoresis pattern, the existence of globulin and alcohol soluble protein, make the wheat glutenin subunit, particularly the differentiation of each band of low molecular weight glutenin subunit is extremely not obvious, has had a strong impact on the research and the detection thereof of each subunit of wheat glutenin.Only under the situation of fully picking out albumin, globulin and alcohol soluble protein, and break disulfide bond between each subunit, could distinguish high and low each subunit of molecular weight glutenin clearly, for the genetic research and the Breeding Application of wheat quality lays the foundation.
Summary of the invention
The object of the present invention is to provide a kind of method of extracting the high low molecular weight glutenin subunit of wheat, by adding the alcohol soluble protein in the isopropyl alcohol removal wheat gluten, add NaI and remove globulin, and water is removed albumin, add DDT, VP and mercaptoethanol and fully break the combination of disulfide bond between the subunit, thereby make each subunit be in released state, for SDS-PAGE lays a good foundation.
Technical scheme of the present invention is achieved in that 1, the method for separating and analyzing of the high and low molecular weight glutenin subunit of wheat, it is characterized in that taking following steps to finish:
A, get the broken 0.5ml of the putting into ep pipe of 1/3 einkorn seed folder, add the extract I that contains isopropyl alcohol and NaI, alcohol soluble protein, globulin and the albumin in the wheat gluten removed in centrifuging;
B, adding contain the extract II of solution B and dithiothreitol (DTT), centrifuging;
C, adding contain the extract III of solution B and 4-vinylpridine;
D, adding glutenin extract IV;
E, utilize sodium dodecyl sulfate-polyacrylamide gel electrophoresis to measure.
The method for separating and analyzing of the high and low molecular weight glutenin subunit of described wheat, the described solution I of steps A are that 0.3MNaI is dissolved in 7.5% isopropyl alcohol.
The method for separating and analyzing of the high and low molecular weight glutenin subunit of described wheat, the described separation method of steps A are to add 200 μ l extract I in the ep pipe, in 65 ℃ of water-bath 1-2 hours, and the centrifugal 3-5 of 10000rpm minute, abandoning supernatant.
The method for separating and analyzing of the high and low molecular weight glutenin subunit of described wheat, the compound method of the described extract II of step B is 0.04M 2-amino-2 methylol-1, ammediol is dissolved in 25% isopropyl alcohol, regulate pH and be 8.0 and obtain solution B, the 10ml solution B adds the 0.1g lauryl sodium sulfate, and the 200mg dithiothreitol (DTT) obtains extract II.
The method for separating and analyzing of the high and low molecular weight glutenin subunit of described wheat, the described separation method of step B are to add 50 μ l extract II in the sediment that steps A obtains, suspend, and 65 ℃ of water-baths 2 hours, centrifugal 3 minutes of 10000rpm gets supernatant.
The method for separating and analyzing of the high and low molecular weight glutenin subunit of described wheat, the compound method of the described extract III of step C is: the 10ml solution B adds the 0.1g lauryl sodium sulfate, and 140 μ l 4-vinylpridines obtain extract III.
The method for separating and analyzing of the high and low molecular weight glutenin subunit of described wheat, the described separation method of step C are to add 50 μ l extract III in the supernatant that step B obtains, 65 ℃ of water-baths 1 hour.
The method for separating and analyzing of the high and low molecular weight glutenin subunit of described wheat, the compound method of the described extract IV of step D is: 2% lauryl sodium sulfate, 5% mercaptoethanol, 20% glycerine, 0.2% bromophenol blue, 2-amino-2-methylol-1 of 7.57g, ammediol, water surplus, pH are 6.8.
The method for separating and analyzing of the high and low molecular weight glutenin subunit of described wheat, the described separation method of step D is a solution of getting step C, add 50 μ l extract IV, mixing, left standstill 30 minutes, 100 ℃ of water-baths 5 minutes, cooling back gained solution is the sample solution that contains the high and low molecular weight glutenin of wheat, but refrigerator is preserved or is directly carried out electrophoretic analysis.
Technical progress effect of the present invention shows: by adding the alcohol soluble protein in the certain density isopropyl alcohol removal wheat gluten, add NaI and remove globulin, and water is removed albumin, add dithiothreitol (DTT), 4-vinylpridine and mercaptoethanol centrifuging successively and fully break the combination of disulfide bond between the subunit, thereby make each subunit be in released state, lay a good foundation for utilizing lauryl sodium sulfate one polyacrylamide gel electrophoresis.
Description of drawings
Fig. 1 is that the prior art compartment analysis is analyzed the high and low molecular weight glutenin subunit of wheat electrophoresis photo
Fig. 2 is among the high and low molecular weight glutenin subunit of the method for separating and analyzing wheat of the present invention electrophoresis photo figure: 1, high molecular weight glutenin subunit of wheat through 2, wheat low molecular weight glutenin subunit
Embodiment
One, the preparation of testing liquid
1, the preparation of extract I: add 45 gram NaI in every liter 7.5% isopropyl alcohol, obtain 0.3MNaI solution;
2, the preparation of extract B: every liter 25% isopropyl alcohol adds 0.48 gram 2-amino-2 methylol-1, and ammediol obtains 0.04M Tris solution, regulates pH=8.0;
3, the preparation of extract II: the 10ml solution B adds 0.1g lauryl sodium sulfate (SDS), is adding 200mg dithiothreitol (DTT) (DTT);
4, the preparation of extract III: the 10ml solution B adds 0.1g lauryl sodium sulfate (SDS), adds 140 μ l 4-vinylpridines (VP) again;
5, the preparation of extract IV: 2% lauryl sodium sulfate (SDS), 5%% mercaptoethanol, 20% glycerine, 0.2% bromophenol blue, 7.57g 2-amino-2-methylol-1, ammediol (Tris), water surplus.Regulate pH=6.8
Two, specimen preparation
1, gets the broken 0.5ml of the putting into ep pipe of 1/3 einkorn seed folder, add 200 μ l and get liquid I;
2,65 ℃ water-bath 1-2 hour, the centrifugal 3-5 of 10000rpm minute, abandon supernatant;
3, add 50 μ l extract II, suspend, 65 ℃ of water-baths 2 hours;
4,10000rpm is centrifugal 3 minutes, gets supernatant and adds 50 μ l extract III, 65 ℃ of water-baths 1 hour;
5, add 50 μ l extract IV, mixing left standstill 30 minutes;
6,100 ℃ of water-baths are 5 minutes, and cooling back gained solution is the sample solution that contains the high and low molecular weight glutenin of wheat, can directly carry out next step electrophoresis, carries out the qualitative examination of high and low molecular weight glutenin subunit.
By the high and low molecular weight glutenin subunit of contrast Fig. 1, Fig. 2 electrophoresis photo, the method for separating and analyzing that the present invention's employing is described is under the situation of fully picking out albumin, globulin and alcohol soluble protein, and break the disulfide bond between each subunit, could distinguish high and low each subunit of molecular weight glutenin clearly, for the genetic research and the Breeding Application of wheat quality lays the foundation.
Listed examples of the present invention is intended to set forth further high and low molecular weight glutenin subunit method for separating and analyzing in the wheat, and scope of the present invention is not constituted any restriction.
Claims (1)
1, the method for separating and analyzing of the high and low molecular weight glutenin subunit of wheat is characterized in that taking following steps to finish:
A, get the broken 0.5ml of the putting into ep pipe of 1/3 einkorn seed folder, add the extract I that contains isopropyl alcohol and NaI, alcohol soluble protein, globulin and the albumin in the wheat gluten removed in centrifuging;
The compound method of described extract I is to add 45 gram NaI in every liter 7.5% isopropyl alcohol, obtains 0.3MNaI solution;
The described centrifuging of steps A is to add 200 μ l extract I in the ep pipe, in 65 ℃ of water-bath 1-2 hours, and the centrifugal 3-5 of 10000rpm minute, abandoning supernatant;
B, adding contain the extract II of solution B and dithiothreitol (DTT), centrifuging;
The compound method of described extract II is that every liter 25% isopropyl alcohol adds 0.48 gram 2-amino-2 methylol-1, ammediol obtains 0.04MTris solution, regulates pH and is 8.0 and obtain solution B, the 10ml solution B adds the 0.1g lauryl sodium sulfate, and the 200mg dithiothreitol (DTT) obtains extract II;
The described centrifuging of step B is to add 50 μ l extract II in the sediment that steps A obtains, suspends, and 65 ℃ of water-baths 2 hours, centrifugal 3 minutes of 10000rpm gets supernatant;
C, adding contain the extract III of solution B and 4-vinylpridine;
The compound method of described extract III is: the 10ml solution B adds the 0.1g lauryl sodium sulfate, and 140 μ l 4-vinylpridines obtain extract III;
In the supernatant that step B obtains, add 50 μ l extract III, 65 ℃ of water-baths 1 hour;
D, adding glutenin extract IV;
The compound method of described extract IV is: 2% lauryl sodium sulfate, and 5% mercaptoethanol, 20% glycerine, 0.2% bromophenol blue, 2-amino-2-methylol-1 of 7.57g, ammediol, water surplus, pH are 6.8;
Get the solution of step C, add 50 μ l extract IV, mixing left standstill 30 minutes, 100 ℃ of water-baths 5 minutes, and cooling back gained solution is the sample solution that contains the high and low molecular weight glutenin of wheat;
E, utilize sodium dodecyl sulfate-polyacrylamide gel electrophoresis to measure.
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| CNB2005100482147A CN100494962C (en) | 2005-12-26 | 2005-12-26 | Wheat high-low molecular weight glutenin subunit separating analytical method |
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| CNB2005100482147A CN100494962C (en) | 2005-12-26 | 2005-12-26 | Wheat high-low molecular weight glutenin subunit separating analytical method |
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| CN100494962C true CN100494962C (en) | 2009-06-03 |
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Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101696238B (en) * | 2009-10-27 | 2012-01-11 | 广东省农业科学院作物研究所 | Plant total protein extracting solution and application thereof |
| CN102998461B (en) * | 2012-11-19 | 2014-08-13 | 杭州市质量技术监督检测院 | Quick qualitative detection reagent for wheat globulin in milk and protein drinks and preparation method thereof |
| CN105021686A (en) * | 2015-07-07 | 2015-11-04 | 青岛农业大学 | Electrophoresis method for simultaneous detection of high molecular weight and low molecular weight glutenin subunits |
| CN105949294A (en) * | 2016-07-15 | 2016-09-21 | 朱广双 | Novel method for one-time complete extraction of wheat glutenin subunits |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US2861061A (en) * | 1956-11-27 | 1958-11-18 | Hercules Powder Co Ltd | Method for treating gluten |
| EP0361838B1 (en) * | 1988-09-30 | 1995-02-22 | Biotechnology and Biological Sciences Research Council | Determining cereal quality |
| US5610277A (en) * | 1995-09-11 | 1997-03-11 | Midwest Grain Products | Alcohol-free wet extraction of gluten dough into gliadin and glutenin |
| CN1541068A (en) * | 2001-08-10 | 2004-10-27 | ����¬ķŷ��˾ | Method for prepn. of gliadin-and glutenin-rich fractions out of gluten in aqueous medium and in presence of acid |
-
2005
- 2005-12-26 CN CNB2005100482147A patent/CN100494962C/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US2861061A (en) * | 1956-11-27 | 1958-11-18 | Hercules Powder Co Ltd | Method for treating gluten |
| EP0361838B1 (en) * | 1988-09-30 | 1995-02-22 | Biotechnology and Biological Sciences Research Council | Determining cereal quality |
| US5610277A (en) * | 1995-09-11 | 1997-03-11 | Midwest Grain Products | Alcohol-free wet extraction of gluten dough into gliadin and glutenin |
| CN1541068A (en) * | 2001-08-10 | 2004-10-27 | ����¬ķŷ��˾ | Method for prepn. of gliadin-and glutenin-rich fractions out of gluten in aqueous medium and in presence of acid |
Non-Patent Citations (2)
| Title |
|---|
| 小麦谷蛋白亚基的快速提取分离及SDS-PAGE分析. 段淑娥，赵文明.陕西师范大学学报（自然科学版），第32卷第1期. 2004 |
| 小麦谷蛋白亚基的快速提取分离及SDS-PAGE分析. 段淑娥，赵文明.陕西师范大学学报（自然科学版），第32卷第1期. 2004 * |
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