CN103278547B - Method for detecting Kunitz trypsin inhibitor deletion in soybean seed - Google Patents
Method for detecting Kunitz trypsin inhibitor deletion in soybean seed Download PDFInfo
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- CN103278547B CN103278547B CN201310141334.6A CN201310141334A CN103278547B CN 103278547 B CN103278547 B CN 103278547B CN 201310141334 A CN201310141334 A CN 201310141334A CN 103278547 B CN103278547 B CN 103278547B
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Abstract
The invention relates to a method for detecting Kunitz trypsin inhibitor deletion in a soybean seed. The method comprises a first step of extracting a small amount of protein from a single soybean seed; a second step of preparing a discontinuous non-denaturing polyacrylamide gel; and a third step of analyzing the deletion situations of the Kunitz trypsin inhibitor by using non-denaturing polyacrylamide gel electrophoresis and scan, wherein the discontinuous non-denaturing polyacrylamide gel is composed of 12% of a separation gel and 4% of a stacking gel. A rapid and simple protein extraction method and the non-denaturing high concentration polyacrylamide gel electrophoresis (Native-PAGE) technology are adopted to detect whether the Kunitz trypsin inhibitor in cultivating offspring soybean seeds is deleted or not. A breeding process is accelerated, antinutritional factors are decreased and protein utilization is improved, by using a biological assisted breeding method.
Description
Technical field
The present invention relates to the detection of Kunitz trypsin inhibitor, be specifically related to a kind of method detecting Kunitz trypsin inhibitor disappearance in soya seeds, belong to biological technical field.
Background technology
Soybean contains rich in protein, essential amino acids content that it has is balanced, water-soluble protein content is high, easily absorbed and the advantage such as utilization, but be unfavorable for that the mankind eat and affect the protein ingredient of food quality containing trypsin inhibitor and lipoxidase etc. in soybean, the nutritive value of soybean protein and flavour of food products are affected.Wherein, trypsin inhibitor is ANFs main in soybean, the trypsin inhibitor had been found that now has 7-10 kind, but study and comparatively detailed only have 2 kinds, i.e. Kunitz trypsin inhibitor (KTI) and Bouman-Birk trypsin inhibitor (BBI).The content of trypsin inhibitor in soybean is about 2%.
KTI by Kunitz in 1945 first from soybean fractional crystallization out name, content is about 1.4%, and molecular weight is about 21kDa, main suppresses the endocrine tryptic activity of animal body.Can be removed by manufacturing process, but both consumes energy, again reduce the utilization factor of albumen.Cultivate low ANFs or the new soybean varieties without ANFs, fundamentally can remove ANFs, for the mankind provide pure natural, without residual hazard, the soybean be of high nutritive value and animal products.Disappearance trypsin inhibitor gene is imported China's soya seeds by Han Fenxia etc., transformation success new soybean varieties, namely the progress not containing KTI(Kunitz trypsin inhibitor in soybean kernel, Luo Yujiao etc., " Chinese biochemical drug impurity Chinese Journal Biochemical Pharmaceutics " the 33rd volume the 3rd phase in 2012).
The present invention is directed to the deletion condition detecting Kunitz trypsin inhibitor in soya seeds how fast and effectively, provide a kind of fast and convenient Protein Extraction method and native polyacrylamide gel electrophoresis (Native-PAGE) technology, detect the Kunitz trypsin inhibitor cultivated in progeny seed whether to lack, utilize biological assistant breeding means, accelerate breeding process, reduce ANFs, improve protein utilization rate.
Summary of the invention
Object of the present invention is for providing a kind of method detecting Kunitz trypsin inhibitor disappearance in soya seeds, and object of the present invention is achieved through the following technical solutions:
(1) albumen in Trace bio-element soybean single seed;
(2) prepare discontinuous non-denaturing polyacrylamide gel, described discontinuous non-denaturing polyacrylamide gel is separation gel and the 4% concentrated glue of 12%;
(3) with this native polyacrylamide gel electrophoresis and scanning analysis Kunitz trypsin inhibitor deletion condition.
Concrete steps are:
1, the albumen in Trace bio-element soybean single seed, concrete operation step is as follows:
In the opposition side of soybean plumular axis, seed fine powder 6-8mg is cut with blade, wherein not containing seed coat, put into the centrifuge tube of 1.5mL, add 300 μ L Extraction buffers, extract the concentration >=20mg/mL of protein sample, stir with oscillator, ultrasonic extraction 5min, with 4 DEG C of centrifugal 10min of supercentrifuge 8000rpm, gets supernatant 20 μ L for Native-PAGE electrophoretic analysis; Described Extraction buffer is 0.1mol/LTris-HCl, pH8.0, and including massfraction is the bromophenol blue of 0.1% and the glycerine of 2%.
Further, the concentration extracting albumen is 23mg/mL.
2, discontinuous non-denaturing polyacrylamide gel is prepared
The assembling of glue glass plate:
Preparation glue thickness is that the electrophoresis rubber-making simple glass plate 1 of 1mm overlaps, 1 cover is 2 pieces, specification is 160mm × 160mm × 5mm, with 70% ethanol by the inside surface wiped clean of glass plate and airing, placing thickness in all peripheral inner side of glass plate except the comb nose end of upper end is 1mm sealed silicon adhesive tape, glue-leakage-resistant, each side uses 2 large size binder clip fastening glass panels, is vertically positioned on horizontal experiment table for subsequent use.
Prepare 12% separating gel liquid:
The Tris-HCl of A liquid: 1.5mol/L, pH8.8;
C liquid: 100mL acrylamide solution, wherein containing acrylamide 30g, methylene diacrylamide 0.8g;
P liquid: now with the current massfraction is the ammonium persulfate of 1.5%;
By A liquid 4.5mL, C liquid 7.5mL, P liquid 0.9mL, ultrapure water 5.1mL, join in 100mL Erlenmeyer flask by the sequencing of the last ultrapure water of P liquid again of C liquid after first A liquid, after fully mixing, add TEMED(N, N, N', N'-tetramethylethylenediamine) stoste 12 μ L, once again after mixing, slowly be injected in the glue glass plate gap assembled, distance glass plate upper end stops when being about 5cm, gap size is gel thicknesses, the top in glue face instills distilled water gently and is about 5mL, prevent bubble from entering colloid, make glue face smooth, after room temperature placement 30-40min glue solidifies, outwell top distilled water, stood upside down in desktop, removing residual moisture is for subsequent use.
The concentrated glue of preparation 4%:
The Tris-HCl of B liquid: 0.5mol/L, pH6.8;
E liquid: 4mg lactochrome, adds ultrapure water 100mL and mixes preparation;
By B liquid 1.6mL, C liquid 0.8mL, E liquid 0.3mL, P liquid 0.2mL, after ultrapure water 2.1mL fully mixes, then adds TEMED(N, N, N', N'-tetramethylethylenediamine) 6 μ L mix, be poured into the upper strata of separation gel, insert comb, room temperature places 30-40min, concentrated gelling is dropped back lower comb and binder clip admittedly, rinse out gently with distilled water and solidify incomplete cull, wrap with preservative film, 4 DEG C of inversions (glue glass plate has the side of comb end to place downwards) refrigerate for subsequent use.
3, native polyacrylamide gel electrophoresis and scanning analysis Kunitz trypsin inhibitor, concrete steps are as follows:
Electrophoretic buffer: the dilution of 10 × electrophoretic buffer forms, i.e. 10 × the electrophoretic buffer of 100mL, diluting also constant volume with ultrapure water is the electrophoretic buffer of 1L, 10 × electrophoretic buffer (Tris-Gly) is the Tris-HCl of glycocoll by 0.192mol/L and 0.025mol/L, pH8.3, i.e. 30.35g Tris and 144g glycocoll, after being settled to 1L with ultrapure water, pH is adjusted to be 8.3,4 DEG C of preservations with hydrochloric acid;
Dyeing liquor: Kao Masi light blue G-2501.5g, adds 440mL and analyzes ethanol, 60mL acetic acid, is settled to 1L mixing formulated with ultrapure water;
Destainer: 200mL methyl alcohol, 50mL acetic acid, is settled to 1L with ultrapure water formulated;
The electrophoretic buffer prepared is added Vertial electrophorestic tank, is about 1cm place to distance vertical electrophoresis groove edge, mounting glass plate, on Vertial electrophorestic tank, notices that the bottom surface of glue in glue glass plate does not enter bubble; After groove is fixed on glass plate, in the middle of two pieces of glass plates, pour electrophoretic buffer into, make liquid level not have the upper end indentation, there of glass plate.Extract the protein sample 20 μ L extracted with 50 μ L micro syringes, slowly inject each swimming lane, each swimming lane sample.Deposition condition: be voltage 100V, electrophoresis time 50-70min, to be instructed dose by after concentrated glue, voltage is risen to 180V, electrophoresis time 3-5 hour, indicator is close to bottom glass plate, stop electrophoresis, after taking off glass plate, with icking tool, colloid is taken off from glass plate gently, put into 1L dyeing liquor, 40-60rmp jog dyes 1 hour, colloid takes out from dyeing liquor, put into 1L destainer subsequently, the decolouring in 4-6 hour of 40-60rmp jog, colloid after decolouring is by intelligent chemiluminescence imaging system (ChemiDoc XRS+, Bio-Rad) in, Protein analysis software scans, imaging, preserve, by gained graphical analysis examine the deletion condition of Kunitz trypsin inhibitor in seed, if there is the band of 21KD, then illustrate containing Kunitz trypsin inhibitor, otherwise, Kunitz trypsin inhibitor disappearance is then described.
Accompanying drawing explanation
Fig. 1 is that Native-PAGE detects filial generation F2 for Kunitz trypsin inhibitor gel image scanning figure in seed; Wherein,
1st swimming lane is Kunitz trypsin inhibitor standard specimen (article No. is T2327, Sigma);
2nd swimming lane is Huang 16 in the male parent of disappearance Kunitz trypsin inhibitor;
3rd swimming lane is maternal conjunction rich 50;
All the other swimming lanes are for hybridization F2 is for seed 10 samples;
Fig. 2 is that Native-PAGE detects filial generation F2 generation and closes rich 50 and backcross the F2 that obtains for Kunitz trypsin inhibitor gel image scanning figure in seed; Wherein, 1-12 swimming lane is backcross in hybridization F2 generation to obtain 12 samples of F2 for seed.
Embodiment
Further describe the present invention below in conjunction with specific embodiment, advantage and disadvantage of the present invention will be more clear along with description.But embodiment is only exemplary, does not form any restriction to scope of the present invention.It will be understood by those skilled in the art that and can modify to the details of technical solution of the present invention and form or replace down without departing from the spirit and scope of the present invention, but these amendments and replacement all fall within the scope of protection of the present invention.
Embodiment 1
Material prepares: utilize the high yield and high quality soybean varieties of the main cultivation in Heilongjiang Province to close rich 50 as maternal, middle yellow 16 of Chinese Academy of Agricultural Sciences's cultivation carries out sexual hybridization as male parent, wherein, one of middle parent of yellow 16 is U.S. soybean kind L81-4590, L81-4590 is the fine quality of disappearance Kunitz trypsin inhibitor, middle yellow 16 disappearance Kunitz trypsin inhibitors.Involutory rich 50 breed with middle yellow 16 filial generation F1 generation seeds, namely normally plant, obtain F2 for hybrid seed, utilize non-denaturing polyacrylamide gel to carry out determination and analysis to F2 for Kunitz trypsin inhibitor in hybrid seed.
Close rich 50 and agents useful for same be commercial, middle yellow 16 introduced varieties that are Chinese Academy of Agricultural Sciences's crop investigations.
Concrete detection method is as follows:
1, Trace bio-element F2 is for the albumen in soybean single seed, and concrete operation step is as follows:
In the opposition side of F2 for hybrid seed plumular axis, seed fine powder 6mg is cut with blade, wherein not containing seed coat, put into the centrifuge tube of 1.5mL, add 300 μ L Extraction buffers, the concentration extracting protein sample is 20mg/mL, stir with oscillator, ultrasonic extraction 5min, with 4 DEG C of centrifugal 10min of supercentrifuge 8000rpm, gets supernatant 20 μ L for Native-PAGE electrophoretic analysis; Described Extraction buffer is 0.1mol/LTris-HCl, pH8.0, and including massfraction is the bromophenol blue of 0.1% and the glycerine of 2%.
2, discontinuous non-denaturing polyacrylamide gel is prepared
The assembling of glue glass plate:
Preparation glue thickness is that the electrophoresis rubber-making simple glass plate 1 of 1mm overlaps, 1 cover is 2 pieces, specification is 160mm × 160mm × 5mm, with the inside surface wiped clean airing of 70% ethanol by glass plate, placing thickness in the peripheral inner side of glass plate except the comb nose end of upper end is 1mm sealed silicon adhesive tape, for glue-leakage-resistant, each side 2 large size binder clips are used for fastening glass panels, are vertically positioned on horizontal experiment table for subsequent use.
Prepare 12% separating gel liquid:
The Tris-HCl of A liquid: 1.5mol/L, pH8.8;
C liquid: 100mL acrylamide solution, wherein containing acrylamide 30g, methylene diacrylamide 0.8g;
P liquid: now with the current massfraction is the ammonium persulfate of 1.5%;
By A liquid 4.5mL, C liquid 7.5mL, P liquid 0.9mL, ultrapure water 5.1mL, join in 100mL Erlenmeyer flask by the sequencing of the last ultrapure water of P liquid again of C liquid after first A liquid, after fully mixing, add TEMED stoste 12 μ L, once again after mixing, slowly be injected in the glue glass plate gap assembled, distance glass plate upper end stops when being about 5cm, gap size is gel thicknesses, the top in glue face instills distilled water gently and is about 5mL, prevent bubble from entering colloid, make glue face smooth, after room temperature placement 35min glue solidifies, outwell top distilled water, stood upside down in desktop, removing residual moisture is for subsequent use.
The concentrated glue of preparation 4%:
The Tris-HCl of B liquid: 0.5mol/L, pH6.8;
E liquid: 4mg lactochrome, adds ultrapure water 100mL and mixes preparation;
By B liquid 1.6mL, C liquid 0.8mL, E liquid 0.3mL, P liquid 0.2mL, after ultrapure water 2.1mL fully mixes, then add TEMED6 μ L and mix, be poured into the upper strata of separation gel, insert comb, room temperature places 35min, and concentrated gelling is dropped back lower comb and binder clip admittedly, rinses out gently solidify incomplete cull with distilled water, wrap with preservative film, 4 DEG C of inversions (glue glass plate has the side of comb end to place downwards) refrigerate for subsequent use.
3, native polyacrylamide gel electrophoresis and scanning analysis Kunitz trypsin inhibitor, concrete steps are as follows:
Electrophoretic buffer: the dilution of 10 × electrophoretic buffer forms, i.e. 10 × the electrophoretic buffer of 100mL, diluting also constant volume with ultrapure water is the electrophoretic buffer of 1L, 10 × electrophoretic buffer (Tris-Gly) is the Tris-HCl of glycocoll by 0.192mol/L and 0.025mol/L, pH8.3, i.e. 30.35g Tris and 144g glycocoll, after being settled to 1L with ultrapure water, pH is adjusted to be 8.3,4 DEG C of preservations with hydrochloric acid;
Dyeing liquor: Kao Masi light blue G-2501.5g, adds 440mL and analyzes ethanol, 60mL acetic acid, is settled to 1L mixing formulated with ultrapure water;
Destainer: 200mL methyl alcohol, 50mL acetic acid, is settled to 1L with ultrapure water formulated;
The electrophoretic buffer prepared is added Vertial electrophorestic tank, is about 1cm place to distance vertical electrophoresis groove edge, mounting glass plate, on Vertial electrophorestic tank, notices that the bottom surface of glue in glue glass plate does not enter bubble; After groove is fixed on glass plate, in the middle of two pieces of glass plates, pour electrophoretic buffer into, make liquid level not have the upper end indentation, there of glass plate.Extract the protein sample 20 μ L carried with 50 μ L micro syringes, slowly inject each swimming lane, each swimming lane sample.Deposition condition: be voltage 100V, electrophoresis time 1 hour 10min, to be instructed dose by after concentrated glue, voltage is risen to 180V, indicator is close to bottom glass plate, electrophoresis time 4 hours 20min, stop electrophoresis, after taking off glass plate, with icking tool, colloid is taken off from glass plate gently, put into 1L dyeing liquor, 50rmp jog dyes 1 hour, colloid takes out from dyeing liquor, put into 1L destainer subsequently, the decolouring in 5 hours of 50rmp jog, colloid after decolouring is by intelligent chemiluminescence imaging system (ChemiDoc XRS+, Bio-Rad) in, Protein analysis software scans, imaging, as shown in Figure 1, result shows, sample disappearance Kunitz trypsin inhibitor in 6th swimming lane.
Embodiment 2
Material prepares: utilize the high yield and high quality soybean varieties of the main cultivation in Heilongjiang Province to close rich 50 as maternal, middle yellow 16 of Chinese Academy of Agricultural Sciences's cultivation carries out sexual hybridization as male parent, wherein, one of middle parent of yellow 16 is U.S. soybean kind L81-4590, L81-4590 is the fine quality of disappearance Kunitz trypsin inhibitor, middle yellow 16 disappearance Kunitz trypsin inhibitors.Involutory rich 50 breed with middle yellow 16 filial generation F1 generation seeds, i.e. normal plantation, obtain F2 for hybrid seed, utilize native polyacrylamide gel electrophoresis to carry out Kunitz trypsin inhibitor disappearance to F2 for hybrid seed to detect, the F2 choosing disappearance Kunitz trypsin inhibitor is the male parent that backcrosses for seed, close rich 50 for the female parent that backcrosses, backcross progeny F1 generation seed is bred, i.e. normal plantation, obtain F2 for backcrossing seed, native polyacrylamide gel electrophoresis is utilized to carry out determination and analysis to F2 for Kunitz trypsin inhibitor in the seed that backcrosses.
Close rich 50 and agents useful for same be commercial, middle yellow 16 introduced varieties that are Chinese Academy of Agricultural Sciences's crop investigations.
Concrete detection method is as follows:
1, the albumen in Trace bio-element soybean single seed, concrete operation step is as follows:
In the opposition side of F2 for the seed plumular axis that backcrosses, seed fine powder 8mg is cut with blade, wherein not containing seed coat, put into the centrifuge tube of 1.5mL, add 300 μ L Extraction buffers, extract the concentration 26mg/mL of protein sample, stir with oscillator, ultrasonic extraction 5min, with 4 DEG C of centrifugal 10min of supercentrifuge 8000rpm, gets supernatant 20 μ L for Native-PAGE electrophoretic analysis; Described Extraction buffer is 0.1mol/LTris-HCl, pH8.0, and including massfraction is the bromophenol blue of 0.1% and the glycerine of 2%.
2, discontinuous non-denaturing polyacrylamide gel is prepared
The assembling of glue glass plate:
Preparation glue thickness is that the electrophoresis rubber-making simple glass plate 1 of 1mm overlaps, 1 cover is 2 pieces, specification is 160mm × 160mm × 5mm, with the inside surface wiped clean airing of 70% ethanol by glass plate, placing thickness in the peripheral inner side of glass plate except the comb nose end of upper end is 1mm sealed silicon adhesive tape, for glue-leakage-resistant, each side 2 large size binder clips are used for fastening glass panels, are vertically positioned on horizontal experiment table for subsequent use.
Prepare 12% separating gel liquid:
The Tris-HCl of A liquid: 1.5mol/L, pH8.8;
C liquid: 100mL acrylamide solution, wherein containing acrylamide 30g, methylene diacrylamide 0.8g;
P liquid: now with the current massfraction is the ammonium persulfate of 1.5%;
By A liquid 4.5mL, C liquid 7.5mL, P liquid 0.9mL, ultrapure water 5.1mL, join in 100mL Erlenmeyer flask by the sequencing of the last ultrapure water of P liquid again of C liquid after first A liquid, after fully mixing, add TEMED stoste 12 μ L, once again after mixing, slowly be injected in the glue glass plate gap assembled, distance glass plate upper end stops when being about 5cm, gap size is gel thicknesses, the top in glue face instills distilled water gently and is about 5mL, prevent bubble from entering colloid, make glue face smooth, after room temperature placement 40min glue solidifies, outwell top distilled water, stood upside down in desktop, removing residual moisture is for subsequent use.
The concentrated glue of preparation 4%:
The Tris-HCl of B liquid: 0.5mol/L, pH6.8;
E liquid: 4mg lactochrome, adds ultrapure water 100mL and mixes preparation;
By B liquid 1.6mL, C liquid 0.8mL, E liquid 0.3mL, P liquid 0.2mL, after ultrapure water 2.1mL fully mixes, then add TEMED6 μ L and mix, be poured into the upper strata of separation gel, insert comb, room temperature places 40min, and concentrated gelling is dropped back lower comb and binder clip admittedly, rinses out gently solidify incomplete cull with distilled water, wrap with preservative film, 4 DEG C of inversions (glue glass plate has the side of comb end to place downwards) refrigerate for subsequent use.
3, native polyacrylamide gel electrophoresis and scanning analysis Kunitz trypsin inhibitor, concrete steps are as follows:
Electrophoretic buffer: the dilution of 10 × electrophoretic buffer forms, i.e. 10 × the electrophoretic buffer of 100mL, diluting also constant volume with ultrapure water is the electrophoretic buffer of 1L, 10 × electrophoretic buffer (Tris-Gly) is the Tris-HCl of glycocoll by 0.192mol/L and 0.025mol/L, pH8.3, i.e. 30.35g Tris and 144g glycocoll, after being settled to 1L with ultrapure water, pH is adjusted to be 8.3,4 DEG C of preservations with hydrochloric acid;
Dyeing liquor: Kao Masi light blue G-2501.5g, adds 440mL and analyzes ethanol, 60mL acetic acid, is settled to 1L mixing formulated with ultrapure water;
Destainer: 200mL methyl alcohol, 50mL acetic acid, is settled to 1L with ultrapure water formulated;
The electrophoretic buffer prepared is added Vertial electrophorestic tank, is about 1cm place to distance vertical electrophoresis groove edge, mounting glass plate, on Vertial electrophorestic tank, notices that the bottom surface of glue in glue glass plate does not enter bubble; After groove is fixed on glass plate, in the middle of two pieces of glass plates, pour electrophoretic buffer into, make liquid level not have the upper end indentation, there of glass plate.Extract the protein sample 20 μ L carried with 50 μ L micro syringes, slowly inject each swimming lane, each swimming lane sample.Deposition condition: be voltage 100V, electrophoresis time 1 hour, to be instructed dose by after concentrated glue, voltage is risen to 180V, electrophoresis time 3 hours 50min, indicator is close to bottom glass plate, stop electrophoresis, after taking off glass plate, with icking tool, colloid is taken off from glass plate gently, put into 1L dyeing liquor, 40rmp jog dyes 1 hour, colloid takes out from dyeing liquor, put into 1L destainer subsequently, the decolouring in 5 hours of 40rmp jog, colloid after decolouring is by intelligent chemiluminescence imaging system (ChemiDoc XRS+, Bio-Rad) in, Protein analysis software scans, imaging, as shown in Figure 2, result shows, sample disappearance Kunitz trypsin inhibitor in 1-7 swimming lane.
Claims (4)
1. detect a method for Kunitz trypsin inhibitor disappearance in soya seeds, it is characterized in that, comprise the following steps:
(1) albumen in Trace bio-element soybean single seed;
In the opposition side of soybean plumular axis, seed fine powder 6-8mg is cut with blade, wherein not containing seed coat, put into the centrifuge tube of 1.5mL, add 300 μ L Extraction buffers, extract the concentration >=20mg/mL of protein sample, stir with oscillator, ultrasonic extraction 5min, with 4 DEG C of centrifugal 10min of supercentrifuge 8000rpm, gets supernatant 20 μ L for Native-PAGE electrophoretic analysis;
Described Extraction buffer is 0.1mol/LTris-HCl, pH8.0, and including massfraction is the bromophenol blue of 0.1% and the glycerine of 2%;
(2) prepare discontinuous non-denaturing polyacrylamide gel, described discontinuous non-denaturing polyacrylamide gel is separation gel and the 4% concentrated glue of 12%;
The assembling of glue glass plate:
Preparation glue thickness is that the electrophoresis rubber-making simple glass plate 1 of 1mm overlaps, 1 cover is 2 pieces, specification is 160mm × 160mm × 5mm, with 70% ethanol by the inside surface wiped clean of glass plate and airing, placing thickness in all peripheral inner side of glass plate except the comb nose end of upper end is 1mm sealed silicon adhesive tape, glue-leakage-resistant, each side uses 2 large size binder clip fastening glass panels, is vertically positioned on horizontal experiment table for subsequent use;
Prepare 12% separating gel liquid:
The Tris-HCl of A liquid: 1.5mol/L, pH8.8;
C liquid: 100mL acrylamide solution, wherein containing acrylamide 30g, methylene diacrylamide 0.8g;
P liquid: now with the current massfraction is the ammonium persulfate of 1.5%;
By A liquid 4.5mL, C liquid 7.5mL, P liquid 0.9mL, ultrapure water 5.1mL, join in 100mL Erlenmeyer flask by the sequencing of the last ultrapure water of P liquid again of C liquid after first A liquid, after fully mixing, add TEMED stoste 12 μ L, once again after mixing, slowly be injected in the glue glass plate gap assembled, stop during distance glass plate upper end 5cm, gap size is gel thicknesses, it is 5mL that the top in glue face instills distilled water gently, prevent bubble from entering colloid, make glue face smooth, after room temperature placement 30-40min glue solidifies, outwell top distilled water, stood upside down in desktop, removing residual moisture is for subsequent use,
The concentrated glue of preparation 4%:
The Tris-HCl of B liquid: 0.5mol/l, pH6.8;
E liquid: 4mg lactochrome, adds ultrapure water 100mL and mixes preparation;
By B liquid 1.6mL, C liquid 0.8mL, E liquid 0.3mL, P liquid 0.2mL, after ultrapure water 2.1mL fully mixes, then add TEMED 6 μ L and mix, be poured into the upper strata of separation gel, insert comb, room temperature places 30-40min, and concentrated gelling is dropped back lower comb and binder clip admittedly, rinses out gently solidify incomplete cull with distilled water, wrap with preservative film, 4 DEG C of inversion refrigerations are for subsequent use;
(3) with this native polyacrylamide gel electrophoresis and scanning analysis Kunitz trypsin inhibitor deletion condition:
Electrophoretic buffer: the dilution of 10 × electrophoretic buffer forms, i.e. 10 × the electrophoretic buffer of 100mL, diluting also constant volume with ultrapure water is the electrophoretic buffer of 1L, 10 × electrophoretic buffer Tris-Gly is the Tris-HCl of glycocoll by 0.192mol/L and 0.025mol/L, pH8.3, i.e. 30.35g Tris and 144g glycocoll, after being settled to 1L with ultrapure water, pH is adjusted to be 8.3,4 DEG C of preservations with hydrochloric acid;
Dyeing liquor: Kao Masi light blue G-2501.5g, adds 440mL and analyzes ethanol, 60mL acetic acid, is settled to 1L mixing formulated with ultrapure water;
Destainer: be 200mL methyl alcohol, 50mL acetic acid, is settled to 1L with ultrapure water formulated;
The electrophoretic buffer prepared is added Vertial electrophorestic tank, and to distance vertical electrophoresis groove edge 1cm place, mounting glass plate, on Vertial electrophorestic tank, notices that the bottom surface of glue in glue glass plate does not enter bubble, after groove is fixed on glass plate, in the middle of two pieces of glass plates, pour electrophoretic buffer into, make liquid level not have the upper end indentation, there of glass plate, extract the protein sample 20 μ L carried with 50 μ L micro syringes, slowly inject each swimming lane, each swimming lane sample, deposition condition: be voltage 100V, electrophoresis time 50-70min, to be instructed dose by after concentrated glue, voltage is risen to 180V, electrophoresis time 3-5 hour, indicator is close to bottom glass plate, stop electrophoresis, after taking off glass plate, with icking tool, colloid is taken off from glass plate gently, put into 1L dyeing liquor, 40-60rmp jog dyes 1 hour, colloid takes out from dyeing liquor, put into 1L destainer subsequently, the decolouring in 4-6 hour of 40-60rmp jog, colloid after decolouring is scanned by Protein analysis software in intelligent chemiluminescence imaging system, imaging, by gained graphical analysis examine the deletion condition of Kunitz trypsin inhibitor in seed, if there is the band of 21KD, then illustrate containing Kunitz trypsin inhibitor, otherwise, Kunitz trypsin inhibitor disappearance is then described.
2. method according to claim 1, is characterized in that, the concentration of described extraction albumen is 23mg/mL.
3. method according to claim 1, is characterized in that, when preparing 12% separating gel liquid, room temperature places 35min, and during preparation 4% concentrated glue, room temperature places 35min.
4. method according to claim 1, is characterized in that, during colloid dyeing, 50rmp jog dyes 1 hour, during colloid decolouring, and 50rmp jog 5 hours.
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