CN107219369A - Avoid the assay method of the fribrillin serine phosphorylation level of signal interference - Google Patents
Avoid the assay method of the fribrillin serine phosphorylation level of signal interference Download PDFInfo
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Abstract
What the present invention was provided avoids the assay method of the fribrillin serine phosphorylation level of signal interference, different fribrillin serine phosphorylation levels can equally be detected in postmortem muscle by protein immunoblotting technology, required muscle samples amount is few, operating process is relatively simple, it is reproducible, detection gained band is clear, and experiment price is low, is a kind of efficient method for determining fribrillin serine phosphorylation level in postmortem muscle.The Biochemical changes of serine phosphorylation fribrillin in ovine skeletal muscle can be researched and analysed using this method, also may extend to the research of other animal muscle Physiology and biochemistries.
Description
Technical field
The invention belongs to biological technical field, more particularly to a kind of fribrillin serine for avoiding signal from disturbing
The assay method of phosphorylation level.
Background technology
Protein phosphorylation modification is the important channel of regulation protein structure and function, at present on protein phosphorylation
The method of level is concentrated mainly on the overall phosphorylation level of measure.Phosphorylation modification is likely to occur in serine, threonine, junket ammonia
On acid, the phosphorylation modification of wherein serine is that quantity is most, is carried out for the Biochemical changes of fribrillin phosphorylation
Research, and be essential in scientific research to the research of animal muscle Physiology and biochemistry based on fribrillin phosphorylation
's.Although also there is the specific antibody for certain amino acid in the prior art, fribrillin species is various, all kinds of
Between content difference it is huge, although mass spectrum can detect the phosphorylation level and phosphorylation site of multiple protein, but single
Detect that sample is few, price is high, data analysis is complicated, and there is presently no for certain fribrillin serine phosphorylation level
Immune blotting detection method.Therefore, one kind can polyprotein identification, detect it is quick, it is low-cost measure fribrillin silk
The method of propylhomoserin phosphorylation level is needed badly.
The content of the invention
It is an object of the invention to solve at least the above and/or defect, and provide at least will be described later excellent
Point.
It is a still further object of the present invention to provide a kind of fribrillin serine phosphorylation water for avoiding signal from disturbing
Flat assay method, is distinguished different fribrillins by different fribrillin contents, molecular weight, signal response
Carry out Western blotting.
A further object of the present invention is to provide a kind of fribrillin serine phosphorylation water for avoiding signal from disturbing
Flat assay method, it is to avoid serine phosphorylation signal is interfered with each other between various fribrillins, by using Diagnosis of Sghistosomiasis
Mark realizes the measure to different fribrillin phosphorylation levels, and a certain kind can not accurately be detected by solving in existing method
The problem of fribrillin, cumbersome, somewhat expensive.
The present invention provides a kind of assay method for the fribrillin serine phosphorylation level for avoiding signal from disturbing, bag
Include following steps：
The protein concentration of fribrillin sample is adjusted to 2 μ g/ μ L；
Albumen marker and fribrillin sample are distinguished into loading to different tracks, electrophoretic voltage uses constant pressure
200V, treats that fribrillin sample bromophenol blue mark is run to apart from glue substrate 0.5mm and stops electrophoresis；
Serine phosphorylation level in fribrillin sample is detected by protein immunoblotting method.
Preferably, the assay method of the described fribrillin serine phosphorylation level for avoiding signal from disturbing
In, stay a blank swimming lane between two swimming lanes of albumen marker and fribrillin.
Preferably, the assay method of the described fribrillin serine phosphorylation level for avoiding signal from disturbing
In,
It is more than or equal to 100KDa target protein for molecular weight, selected resolving gel concentration is 7.5%；
Target protein for molecular weight higher than 40KDa and less than 100KDa, selected resolving gel concentration is 10%；
It is less than or equal to 40KDa target protein for molecular weight, selected resolving gel concentration is 12%；
Preferably, the assay method of the described fribrillin serine phosphorylation level for avoiding signal from disturbing
In,
Albumen marker applied sample amounts are 2 μ L；
It is 2.5 μ g for the higher myoglobulin heavy chain of content in fribrillin, actin applied sample amount, for it
His fribrillin applied sample amount is 10 μ g.
Preferably, the assay method of the described fribrillin serine phosphorylation level for avoiding signal from disturbing
In, detected by protein immunoblotting method in the fribrillin serine phosphorylation horizontal process, with the flesh
The first antibody of fibrillin reaction uses serine phosphorylation monoclonal antibody, and the concentration of the first antibody is 60-80 μ
g/ml。
Preferably, the assay method of the described fribrillin serine phosphorylation level for avoiding signal from disturbing
In, detected by protein immunoblotting method in the fribrillin serine phosphorylation horizontal process, with described the
The secondary antibody of one antibody binding uses sheep anti-mouse igg, and the concentration of the secondary antibody is 0.05-0.2 μ g/mL.
Preferably, the assay method of the described fribrillin serine phosphorylation level for avoiding signal from disturbing
In, in the serine phosphorylation level for the fribrillin sample that the phosphorylation is detected by protein immunoblotting method
During,
It is more than or equal to 100KDa target protein for molecular weight, transferring film condition is semidry method transferring film, 3mA, 4.5min；
For molecular weight higher than 40KDa and less than 100KDa target protein, transferring film condition be semidry method transferring film, 3mA,
3.5min；
It is less than or equal to 40KDa target protein for molecular weight, transferring film condition is semidry method transferring film, 3mA, 2.5min.
Preferably, the assay method of the described fribrillin serine phosphorylation level for avoiding signal from disturbing
In, in the serine phosphorylation level for the fribrillin sample that the phosphorylation is detected by protein immunoblotting method
During, before being mixed with the first antibody, rule along albumen marker pencil levels and extend to albumen marker with
Vacant band between fribrillin, and the band is vertically cut off, by remaining fribrillin part and described first
Antibody mixing is incubated.
Preferably, the assay method of the described fribrillin serine phosphorylation level for avoiding signal from disturbing
In, in the serine phosphorylation level for the fribrillin sample that the phosphorylation is detected by protein immunoblotting method
During,
If the non-myoglobulin heavy chain of target, actin are detected, before being mixed with first antibody, along albumen marker water
Straight snips striping middle-molecular-weihydroxyethyl is more than 220KDa bands, 40-45KDa strip portions, remainder corner cut is marked, with first antibody
Mixing；
If it is myoglobulin heavy chain and actin to detect target, before being mixed with first antibody, along albumen marker water
Part and 34-55KDa strip portions, remainder corner cut is marked below straight snips striping middle-molecular-weihydroxyethyl 180KDa bands.
Preferably, the assay method of the described fribrillin serine phosphorylation level for avoiding signal from disturbing
In, the assay method of the described fribrillin serine phosphorylation level for avoiding signal from disturbing, it is characterised in that described
Fribrillin sample is derived from sheep.
The present invention at least includes following beneficial effect：
What the present invention was provided avoids the assay method of the fribrillin serine phosphorylation level of signal interference, passes through
Protein immunoblotting technology can equally detect different fribrillin serine phosphorylation levels in postmortem muscle,
Required muscle samples amount is few, and operating process is relatively simple, reproducible, and detection gained band is clear, and experiment price is low, is one
Plant the method for efficiently determining fribrillin serine phosphorylation level in postmortem muscle.Can be to ovine skeletal muscle using this method
The Biochemical changes of middle serine phosphorylation fribrillin are researched and analysed, and also may extend to the life of other animal muscle physiology
The research of change.
Further advantage, target and the feature of the present invention embodies part by following explanation, and part will also be by this
The research and practice of invention and be understood by the person skilled in the art.
Brief description of the drawings
The assay method for the fribrillin serine phosphorylation level for avoiding signal from disturbing that Fig. 1 provides for the present invention
Embodiment 1 in various concentrations gel myosin image after different time transferring film；
The assay method for the fribrillin serine phosphorylation level for avoiding signal from disturbing that Fig. 2 provides for the present invention
Embodiment 1 in various concentrations gel desmin image after different time transferring film；
The assay method for the fribrillin serine phosphorylation level for avoiding signal from disturbing that Fig. 3 provides for the present invention
Embodiment 1 in various concentrations gel actin image after different time transferring film；
The assay method for the fribrillin serine phosphorylation level for avoiding signal from disturbing that Fig. 4 provides for the present invention
Embodiment 1 in various concentrations gel TnT image after different time transferring film；
The assay method for the fribrillin serine phosphorylation level for avoiding signal from disturbing that Fig. 5 provides for the present invention
Embodiment 2 in albumen marker to exposure signal interference figure；
The assay method for the fribrillin serine phosphorylation level for avoiding signal from disturbing that Fig. 6 provides for the present invention
Embodiment 4 in myoglobulin heavy chain serine phosphorylation exposure image figure；
The assay method for the fribrillin serine phosphorylation level for avoiding signal from disturbing that Fig. 7 provides for the present invention
Embodiment 4 in actin serine phosphorylation exposure image figure；
The assay method for the fribrillin serine phosphorylation level for avoiding signal from disturbing that Fig. 8 provides for the present invention
Embodiment 5 in fribrillin (except myoglobulin heavy chain and actin) serine phosphorylation exposure image figure.
Embodiment
The present invention is described in further detail below in conjunction with the accompanying drawings, to make those skilled in the art with reference to specification text
Word can be implemented according to this.
It should be appreciated that such as " having ", "comprising" and " comprising " term used herein do not allot one or many
The presence or addition of individual other elements or its combination.
The present invention provides a kind of assay method for the fribrillin serine phosphorylation level for avoiding signal from disturbing, bag
Include following steps：
The protein concentration of fribrillin sample is adjusted to 2 μ g/ μ L；
Albumen marker and fribrillin sample are distinguished into loading to different tracks, electrophoretic voltage uses constant pressure
200V, treats that fribrillin sample bromophenol blue mark is run to apart from glue substrate 0.5mm and stops electrophoresis；
Serine phosphorylation level in fribrillin sample is detected by protein immunoblotting method.
In the assay method of the described fribrillin serine phosphorylation level for avoiding signal from disturbing, albumen
A blank swimming lane is stayed between two swimming lanes of marker and fribrillin.
In the assay method of the described fribrillin serine phosphorylation level for avoiding signal from disturbing,
It is more than or equal to 100KDa target protein for molecular weight, selected resolving gel concentration is 7.5%；
Target protein for molecular weight higher than 40KDa and less than 100KDa, selected resolving gel concentration is 10%；
It is less than or equal to 40KDa target protein for molecular weight, selected resolving gel concentration is 12%；
In the assay method of the described fribrillin serine phosphorylation level for avoiding signal from disturbing, albumen
Marker applied sample amounts are 2 μ L；
It is 2.5 μ g for the higher myoglobulin heavy chain of content in fribrillin, actin applied sample amount, for it
His fribrillin applied sample amount is 10 μ g.
In the assay method of the described fribrillin serine phosphorylation level for avoiding signal from disturbing, pass through albumen
Matter western blotting method is detected in the fribrillin serine phosphorylation horizontal process, anti-with the fribrillin
The first antibody answered uses serine phosphorylation monoclonal antibody, and the concentration of the first antibody is 60-80 μ g/ml, or 70 μ g/
ml。
In the assay method of the described fribrillin serine phosphorylation level for avoiding signal from disturbing, pass through albumen
Matter western blotting method is detected in the fribrillin serine phosphorylation horizontal process, is combined with the first antibody
Secondary antibody uses sheep anti-mouse igg, and the concentration of the secondary antibody is 0.05-0.2 μ g/mL.
In the assay method of the described fribrillin serine phosphorylation level for avoiding signal from disturbing, passing through egg
In the serine phosphorylation horizontal process of the fribrillin sample of the white matter western blotting method detection phosphorylation,
It is more than or equal to 100KDa target protein for molecular weight, transferring film condition is semidry method transferring film, 3mA, 4.5min；
For molecular weight higher than 40KDa and less than 100KDa target protein, transferring film condition be semidry method transferring film, 3mA,
3.5min；
It is less than or equal to 40KDa target protein for molecular weight, transferring film condition is semidry method transferring film, 3mA, 2.5min.
In the assay method of the described fribrillin serine phosphorylation level for avoiding signal from disturbing, passing through egg
White matter western blotting method is detected in the serine phosphorylation horizontal process of the fribrillin sample of the phosphorylation, with institute
State before first antibody mixing, rule along albumen marker pencil levels and extend to albumen marker and fribrillin
Between vacant band, and the band is vertically cut off, remaining fribrillin part is mixed into incubation with the first antibody.
In the assay method of the described fribrillin serine phosphorylation level for avoiding signal from disturbing, passing through egg
In the serine phosphorylation horizontal process of the fribrillin sample of the white matter western blotting method detection phosphorylation,
If the non-myoglobulin heavy chain of target, actin are detected, before being mixed with first antibody, along albumen marker water
Straight snips striping middle-molecular-weihydroxyethyl is more than 220KDa bands, 40-45KDa strip portions, remainder corner cut is marked, with first antibody
Mixing；
If it is myoglobulin heavy chain and actin to detect target, before being mixed with first antibody, along albumen marker water
Part and 34-55KDa strip portions, remainder corner cut is marked below straight snips striping middle-molecular-weihydroxyethyl 180KDa bands.
In the assay method of the described fribrillin serine phosphorylation level for avoiding signal from disturbing, described keeps away
Exempt from the assay method of the fribrillin serine phosphorylation level of signal interference, it is characterised in that the muscle fibril egg
White sample is derived from sheep.
The reagent and kit used in the present invention：
2 × reproducibility sample-loading buffer：10%SDS 4mL, glycerine 2mL, 0.5M Tris-HCl pH6.8 2mL, 1M
DTT 2.0mL, bromophenol blue 0.01g：
7.5%th, 10%, 12% separation gel and 4% concentration glue：Bio-Rad#1610181, #1610183, #
1610185；
TBS buffer solutions：Tris 1.211g, NaCl 8.76g, 1mol/L HCl adjust pH to 7.5；
Included in confining liquid：TBS 50ml/L, Tween20 25 μ l/L, BSA 1.5g/L：
TBST1：Add 0.5mL Tween20 per 500mLTBS；
TBST2：Tris 3.0275g, NaCl 4.38g, Tween20 0.5mL, HCl tune pH are settled to 500mL to 7.5；
Protein standards：thermo#26616；
The kit used in ECL development processes is：Bio-Rad#1705061；
The kit that the protein concentration that BCA methods determine extraction sample is used is Thermo#23225；
Following embodiments use the unidirectional electrophoresis systems of Bio-Rad, Bio-Rad semidry method transferring film instrument and Bio-Rad gels
Imaging system.
Embodiment 1
A) the sheep longissimus dorsi muscle fribrillin sample of -80 DEG C of preservations in part is taken, BCA methods determine protein concentration；
B) SDS-PAGE electrophoresis：Using 7.5%, 10%, 12% separation gel and 4% concentration glue, every Kong Jun of gel
10 μ g or 2 μ g protein samples are added, albumen marker applied sample amounts are 2 μ L, for the higher flesh ball of content in fribrillin
Ferritin heavy chain, actin applied sample amount are 2.5 μ g, are 10 μ g for other fribrillin applied sample amounts；It is big for molecular weight
In the target protein equal to 100KDa, using 7.5% separation gel, the mesh for molecular weight higher than 40KDa and less than 100KDa
Albumen is marked, using 10% separation gel, 40KDa target protein is less than or equal to for molecular weight, using 12% separation gel；Electricity
Voltage of swimming uses constant pressure 200V, treats that sample bromophenol blue mark is run to apart from the mm of glue substrate 0.5 and stops electrophoresis；
C) protein immunoblotting (IB)：Electrophoresis unloads offset plate after terminating, and gel is put into transferring film buffer solution and balanced；
PVDF films activate 15s with pure methanol, are put into transferring film buffer solution and balance together with ready transferring film special filter paper；With half-dried
Method transferring film technology so that the protein band on gel is transferred on pvdf membrane；It is more than or equal to 100KDa target for molecular weight
Albumen, transferring film condition is semidry method transferring film, 3mA, 4.5min, the target egg for molecular weight higher than 40KDa and less than 100KDa
In vain, transferring film condition is semidry method transferring film, and 3mA, 3.5min are less than or equal to 40KDa target protein, transferring film condition for molecular weight
For semidry method transferring film, 3mA, 2.5min；After transferring film terminates, PVDF films are washed with TBS buffer solutions three times, each 3min；
Pvdf membrane is put into the hybridization bag cut, adds 5ml confining liquids, at room temperature the concussion closing 2h on shaking table；
First antibody abbreviation primary antibody, primary antibody is incubated：The phosphorylation serine list of Sigma companies is added in 5ml confining liquids
Clonal antibody (article No.：P3430), concentration is 70 μ g/ml；Hybridization bag is changed, by pvdf membrane and 4 DEG C of the primary antibody solution prepared
Night incubation；
Secondary antibody abbreviation secondary antibody, secondary antibody is incubated：Pvdf membrane is washed with TBST1 buffer solutions three times, each 20min；In 5ml
Add the anti-mouse IgG (article No. is A9044) of Sigma companies in confining liquid, concentration is 0.1 μ g/ml, molten as secondary antibody
Liquid is incubated 2h with the concussion of pvdf membrane room temperature；
Pvdf membrane is washed with TBST2 buffer solutions three times, each 10min utilizes the ECL (article No.s of Bio-rid companies afterwards
For) colour developing.
Embodiment 2
A) the sheep longissimus dorsi muscle fribrillin sample of -80 DEG C of preservations in part is taken, BCA methods determine protein concentration；
B) SDS-PAGE electrophoresis：Using 7.5%, 10%, 12% separation gel and 4% concentration glue, every Kong Jun of gel
10 μ g or 2 μ g protein samples are added, albumen marker applied sample amounts are 2 μ L, for the higher flesh ball of content in fribrillin
Ferritin heavy chain, actin applied sample amount are 2.5 μ g, are 10 μ g for other fribrillin applied sample amounts；It is big for molecular weight
In the target protein equal to 100KDa, using 7.5% separation gel, the mesh for molecular weight higher than 40KDa and less than 100KDa
Albumen is marked, using 10% separation gel, 40KDa target protein is less than or equal to for molecular weight, using 12% separation gel；Electricity
Voltage of swimming uses constant pressure 200V, treats that sample bromophenol blue mark is run to apart from the mm of glue substrate 0.5 and stops electrophoresis；
C) protein immunoblotting (IB)：Electrophoresis unloads offset plate after terminating, and gel is put into transferring film buffer solution and balanced；
PVDF films activate 15s with pure methanol, are put into transferring film buffer solution and balance together with ready transferring film special filter paper；With half-dried
Method transferring film technology so that the protein band on gel is transferred on pvdf membrane；It is more than or equal to 100KDa target for molecular weight
Albumen, transferring film condition is semidry method transferring film, 3mA, 4.5min, the target egg for molecular weight higher than 40KDa and less than 100KDa
In vain, transferring film condition is semidry method transferring film, and 3mA, 3.5min are less than or equal to 40KDa target protein, transferring film condition for molecular weight
For semidry method transferring film, 3mA, 2.5min；After transferring film terminates, PVDF films are washed with TBS buffer solutions three times, each 3min；
Pvdf membrane is put into the hybridization bag cut, adds 5ml confining liquids, at room temperature the concussion closing 2h on shaking table；
Albumen marker is cut from pvdf membrane；
First antibody abbreviation primary antibody, primary antibody is incubated：The phosphorylation serine list of Sigma companies is added in 5ml confining liquids
Clonal antibody (article No.：P3430), concentration is 70 μ g/ml；Hybridization bag is changed, albumen marker pvdf membrane and preparation will be cut
Good 4 DEG C of night incubations of primary antibody solution；
Secondary antibody abbreviation secondary antibody, secondary antibody is incubated：Pvdf membrane is washed with TBST1 buffer solutions three times, each 20min；In 5ml
Add the anti-mouse IgG (article No. is A9044) of Sigma companies in confining liquid, concentration is 0.1 μ g/ml, molten as secondary antibody
Liquid is incubated 2h with the concussion of pvdf membrane room temperature；
Pvdf membrane is washed with TBST2 buffer solutions three times, each 10min utilizes the ECL (article No.s of Bio-rid companies afterwards
For) colour developing.
Embodiment 3
A) the sheep longissimus dorsi muscle fribrillin sample of -80 DEG C of preservations in part is taken, BCA methods determine protein concentration；
B) SDS-PAGE electrophoresis：Using 7.5%, 10%, 12% separation gel and 4% concentration glue, every Kong Jun of gel
10 μ g or 2 μ g protein samples are added, albumen marker applied sample amounts are 2 μ L, for the higher flesh ball of content in fribrillin
Ferritin heavy chain, actin applied sample amount are 2.5 μ g, are 10 μ g for other fribrillin applied sample amounts；It is big for molecular weight
In the target protein equal to 100KDa, using 7.5% separation gel, the mesh for molecular weight higher than 40KDa and less than 100KDa
Albumen is marked, using 10% separation gel, 40KDa target protein is less than or equal to for molecular weight, using 12% separation gel；Egg
White marker and sample the blank swimming lane of interval one not loading, electrophoretic voltage use constant pressure 200V, treat that sample bromophenol blue mark is run
Stop electrophoresis to apart from glue substrate 0.5mm；
C) protein immunoblotting (IB)：Electrophoresis unloads offset plate after terminating, and gel is put into transferring film buffer solution and balanced；
PVDF films activate 15s with pure methanol, are put into transferring film buffer solution and balance together with ready transferring film special filter paper；With half-dried
Method transferring film technology so that the protein band on gel is transferred on pvdf membrane；It is more than or equal to 100KDa target for molecular weight
Albumen, transferring film condition is semidry method transferring film, 3mA, 4.5min, the target egg for molecular weight higher than 40KDa and less than 100KDa
In vain, transferring film condition is semidry method transferring film, and 3mA, 3.5min are less than or equal to 40KDa target protein, transferring film condition for molecular weight
For semidry method transferring film, 3mA, 2.5min；After transferring film terminates, PVDF films are washed with TBS buffer solutions three times, each 3min；
Pvdf membrane is put into the hybridization bag cut, adds 5ml confining liquids, at room temperature the concussion closing 2h on shaking table；
Rule along albumen marker levels with pencil, extend to bar vacant between albumen marker and fribrillin
Band, albumen marker is cut from pvdf membrane along in the middle of the blank band between albumen marker and sample；
First antibody abbreviation primary antibody, primary antibody is incubated：The phosphorylation serine list of Sigma companies is added in 5ml confining liquids
Clonal antibody (article No.：P3430), concentration is 70 μ g/ml；Hybridization bag is changed, albumen marker pvdf membrane and preparation will be cut
Good 4 DEG C of night incubations of primary antibody solution；
Secondary antibody abbreviation secondary antibody, secondary antibody is incubated：Pvdf membrane is washed with TBST1 buffer solutions three times, each 20min；In 5ml
Add the anti-mouse IgG (article No. is A9044) of Sigma companies in confining liquid, concentration is 0.1 μ g/ml, molten as secondary antibody
Liquid is incubated 2h with the concussion of pvdf membrane room temperature；
Pvdf membrane is washed with TBST2 buffer solutions three times, each 10min utilizes the ECL (article No.s of Bio-rid companies afterwards
For) colour developing.
Embodiment 4
A) the sheep longissimus dorsi muscle fribrillin sample of -80 DEG C of preservations in part is taken, BCA methods determine protein concentration；
B) SDS-PAGE electrophoresis：Using 7.5%, 10%, 12% separation gel and 4% concentration glue, every Kong Jun of gel
10 μ g or 2 μ g protein samples are added, albumen marker applied sample amounts are 2 μ L, for the higher flesh ball of content in fribrillin
Ferritin heavy chain, actin applied sample amount are 2.5 μ g, are 10 μ g for other fribrillin applied sample amounts；It is big for molecular weight
In the target protein equal to 100KDa, using 7.5% separation gel, the mesh for molecular weight higher than 40KDa and less than 100KDa
Albumen is marked, using 10% separation gel, 40KDa target protein is less than or equal to for molecular weight, using 12% separation gel；Egg
White marker and sample the blank swimming lane of interval one not loading, electrophoretic voltage use constant pressure 200V, treat that sample bromophenol blue mark is run
Stop electrophoresis to apart from glue substrate 0.5mm；
C) protein immunoblotting (IB)：Electrophoresis unloads offset plate after terminating, and gel is put into transferring film buffer solution and balanced；
PVDF films activate 15s with pure methanol, are put into transferring film buffer solution and balance together with ready transferring film special filter paper；With half-dried
Method transferring film technology so that the protein band on gel is transferred on pvdf membrane；It is more than or equal to 100KDa target for molecular weight
Albumen, transferring film condition is semidry method transferring film, 3mA, 4.5min, the target egg for molecular weight higher than 40KDa and less than 100KDa
In vain, transferring film condition is semidry method transferring film, and 3mA, 3.5min are less than or equal to 40KDa target protein, transferring film condition for molecular weight
For semidry method transferring film, 3mA, 2.5min；After transferring film terminates, PVDF films are washed with TBS buffer solutions three times, each 3min；
Pvdf membrane is put into the hybridization bag cut, adds 5ml confining liquids, at room temperature the concussion closing 2h on shaking table；
Rule along albumen marker levels with pencil, extend to bar vacant between albumen marker and fribrillin
Band, albumen marker is cut from pvdf membrane along in the middle of the blank band between albumen marker and sample；
If detection target is actomyosin, actin, before being mixed with first antibody, along albumen marker levels
Part and 34-55KDa strip portions below film middle-molecular-weihydroxyethyl 180KDa bands are cut off, remainder corner cut is marked；
First antibody abbreviation primary antibody, primary antibody is incubated：The phosphorylation serine list of Sigma companies is added in 5ml confining liquids
Clonal antibody (article No.：P3430), concentration is 70 μ g/ml；Hybridization bag is changed, albumen marker pvdf membrane and preparation will be cut
Good 4 DEG C of night incubations of primary antibody solution；
Secondary antibody abbreviation secondary antibody, secondary antibody is incubated：Pvdf membrane is washed with TBST1 buffer solutions three times, each 20min；In 5ml
Add the anti-mouse IgG (article No. is A9044) of Sigma companies in confining liquid, concentration is 0.1 μ g/ml, molten as secondary antibody
Liquid is incubated 2h with the concussion of pvdf membrane room temperature；
Pvdf membrane is washed with TBST2 buffer solutions three times, each 10min utilizes the ECL (article No.s of Bio-rid companies afterwards
For) colour developing.
Embodiment 5
A) the sheep longissimus dorsi muscle fribrillin sample of -80 DEG C of preservations in part is taken, BCA methods determine protein concentration；
B) SDS-PAGE electrophoresis：Using 7.5%, 10%, 12% separation gel and 4% concentration glue, every Kong Jun of gel
10 μ g or 2 μ g protein samples are added, albumen marker applied sample amounts are 2 μ L, for the higher flesh ball of content in fribrillin
Ferritin heavy chain, actin applied sample amount are 2.5 μ g, are 10 μ g for other fribrillin applied sample amounts；It is big for molecular weight
In the target protein equal to 100KDa, using 7.5% separation gel, the mesh for molecular weight higher than 40KDa and less than 100KDa
Albumen is marked, using 10% separation gel, 40KDa target protein is less than or equal to for molecular weight, using 12% separation gel；Egg
White marker and sample the blank swimming lane of interval one not loading, electrophoretic voltage use constant pressure 200V, treat that sample bromophenol blue mark is run
Stop electrophoresis to apart from glue substrate 0.5mm；
C) protein immunoblotting (IB)：Electrophoresis unloads offset plate after terminating, and gel is put into transferring film buffer solution and balanced；
PVDF films activate 15s with pure methanol, are put into transferring film buffer solution and balance together with ready transferring film special filter paper；With half-dried
Method transferring film technology so that the protein band on gel is transferred on pvdf membrane；It is more than or equal to 100KDa target for molecular weight
Albumen, transferring film condition is semidry method transferring film, 3mA, 4.5min, the target egg for molecular weight higher than 40KDa and less than 100KDa
In vain, transferring film condition is semidry method transferring film, and 3mA, 3.5min are less than or equal to 40KDa target protein, transferring film condition for molecular weight
For semidry method transferring film, 3mA, 2.5min；After transferring film terminates, PVDF films are washed with TBS buffer solutions three times, each 3min；
Pvdf membrane is put into the hybridization bag cut, adds 5ml confining liquids, at room temperature the concussion closing 2h on shaking table；
Rule along albumen marker levels with pencil, extend to bar vacant between albumen marker and fribrillin
Band, albumen marker is cut from pvdf membrane along in the middle of the blank band between albumen marker and sample；
If the non-actomyosin of target, actin are detected, before being mixed with first antibody, along albumen marker levels
Film middle-molecular-weihydroxyethyl is cut off more than 220KDa bands, 40-45KDa strip portions, remainder corner cut is marked；
First antibody abbreviation primary antibody, primary antibody is incubated：The phosphorylation serine list of Sigma companies is added in 5ml confining liquids
Clonal antibody (article No.：P3430), concentration is 70 μ g/ml；Hybridization bag is changed, albumen marker pvdf membrane and preparation will be cut
Good 4 DEG C of night incubations of primary antibody solution；
Secondary antibody abbreviation secondary antibody, secondary antibody is incubated：Pvdf membrane is washed with TBST1 buffer solutions three times, each 20min；In 5ml
Add the anti-mouse IgG (article No. is A9044) of Sigma companies in confining liquid, concentration is 0.1 μ g/ml, molten as secondary antibody
Liquid is incubated 2h with the concussion of pvdf membrane room temperature；
Pvdf membrane is washed with TBST2 buffer solutions three times, each 10min utilizes the ECL (article No.s of Bio-rid companies afterwards
For) colour developing.
As shown in Figure 1, Figure 2, Figure 3 and Figure 4, the separation gel for being respectively adopted 7.5%, 10%, 12% carries out gel electrophoresis, mesh
It is designated as myosin and actin, applied sample amount is 2.5 μ g, target is other albumen, applied sample amount is 10 μ g, big for molecular weight
In the target protein equal to 100KDa, transferring film condition is semidry method transferring film, 3mA, 4.5min, for molecular weight higher than 40KDa and
Target protein less than 100KDa, transferring film condition is semidry method transferring film, and 3mA, 3.5min are less than or equal to 40KDa for molecular weight
Target protein, transferring film condition be semidry method transferring film, 3mA, 2.5min.Mhc heavy chain molecule amount is about between 220kDa, flesh
Linear protein molecular weight is about 55kDa, and actin molecular weight is about 43 kDa, and TnT molecular weight is about 37kDa.Using
Above-mentioned condition, it is detected with corresponding antibody test myoglobulin heavy chain, desmin, actin, TnT
Band it is clear, and remained after transferring film on pvdf membrane without target protein.
Fig. 5 is the result of the gained of embodiment 2, as shown in figure 5, using automatic exposure, can only observe clearly albumen
Marker band, illustrates marker there is also serine phosphorylation phenomenon, after overexposure, it may be observed that fribrillin
In some albumen serine phosphorylation modification phenomenon, but albumen marker produces stronger signal to neighbouring lane and disturbs, and cuts
The serine phosphorylation signal for falling to carry out some albumen in overexposure, fribrillin after marker is stronger, but with
Swimming lane adjacent marker still has signal interference phenomenon.
Fig. 6 and Fig. 7 are the results of the gained of embodiment 4, while the method for embodiment 3 is employed, as shown in figure 3, flesh ball egg
Bai Chonglian and actin are two kinds of albumen of content highest in fribrillin, by molecular weight ranges where two albumen
Part is cut before primary antibody incubation from pvdf membrane, can obtain clearly serine phosphorylation band, and by albumen marker
With sample every a swimming lane loading, marker signal interference is successfully eliminated.
Fig. 8 is the result of the gained of embodiment 5, while employ the method for embodiment 3, as shown in figure 8, by myosin weight
After chain and actin are cut from pvdf membrane, both albumen other albumen relatively low to content can be successfully avoided
Signal is disturbed, and can detect a plurality of serine phosphorylation band；And by albumen marker and sample every a swimming lane loading, success
Eliminate marker signal interference；Simultaneously before albumen marker is cut, rule, extended with pencil along albumen marker levels
To band vacant between albumen marker and fribrillin, it can be done with labelled protein molecular weight excluding albumen marker
Also albumen marker effect has been played while disturbing.
Treatment scale described herein is the explanation for simplifying the present invention.To the Phosphorylation and albumen of the present invention
The application of the method such as matter immunoprecipitation and Western blotting, modifications and variations are apparent to one skilled in the art
's.
As described above, according to the present invention, detect that fribrillin serine achieves success in sheep longissimus dorsi muscle, and,
Through testing repeatedly, the inventive method experimental repeatability is good, reliable results, can be fribrillin silk in the animals skeletal muscles such as sheep
The detection research of propylhomoserin phosphorylation provides technical example.
Although embodiment of the present invention is disclosed as above, it is not restricted in specification and embodiment listed
With it can be applied to various suitable the field of the invention completely, can be easily for those skilled in the art
Other modification is realized, therefore under the universal limited without departing substantially from claim and equivalency range, the present invention is not limited
In specific details and shown here as the legend with description.
Claims (10)
1. avoid signal disturb fribrillin serine phosphorylation level assay method, it is characterised in that including with
Lower step：
The protein concentration of fribrillin sample is adjusted to 2 μ g/ μ L；
Albumen marker and fribrillin sample are distinguished into loading to different tracks, electrophoretic voltage uses constant pressure
200V, treats that fribrillin sample bromophenol blue mark is run to apart from glue substrate 0.5mm and stops electrophoresis；
Serine phosphorylation level in fribrillin sample is detected by protein immunoblotting method.
2. the assay method of the fribrillin serine phosphorylation level as claimed in claim 1 for avoiding signal from disturbing,
Characterized in that, staying a blank swimming lane between two swimming lanes of albumen marker and fribrillin.
3. the assay method of the fribrillin serine phosphorylation level as claimed in claim 1 for avoiding signal from disturbing,
Characterized in that,
It is more than or equal to 100KDa target protein for molecular weight, selected resolving gel concentration is 7.5%；
Target protein for molecular weight higher than 40KDa and less than 100KDa, selected resolving gel concentration is 10%；
It is less than or equal to 40KDa target protein for molecular weight, selected resolving gel concentration is 12%.
4. the assay method of the fribrillin serine phosphorylation level as claimed in claim 1 for avoiding signal from disturbing,
Characterized in that,
Albumen marker applied sample amounts are 2 μ L；
It is 2.5 μ g for the higher myoglobulin heavy chain of content in fribrillin, actin applied sample amount, for other fleshes
Fibrillin applied sample amount is 10 μ g.
5. the assay method of the fribrillin serine phosphorylation level as claimed in claim 1 for avoiding signal from disturbing,
Characterized in that, detected by protein immunoblotting method in the fribrillin serine phosphorylation horizontal process,
Serine phosphorylation monoclonal antibody, the concentration of the first antibody are used with the first antibody that the fribrillin reacts
For 60-80 μ g/ml.
6. the assay method of the fribrillin serine phosphorylation level as claimed in claim 5 for avoiding signal from disturbing,
Characterized in that, detected by protein immunoblotting method in the fribrillin serine phosphorylation horizontal process,
The secondary antibody combined with the first antibody uses sheep anti-mouse igg, and the concentration of the secondary antibody is 0.05-0.2 μ g/mL.
7. the assay method of the fribrillin serine phosphorylation level as claimed in claim 3 for avoiding signal from disturbing,
Characterized in that, in the serine phosphorus for the fribrillin sample that the phosphorylation is detected by protein immunoblotting method
During acidifying level,
It is more than or equal to 100KDa target protein for molecular weight, transferring film condition is semidry method transferring film, 3mA, 4.5min；
For molecular weight higher than 40KDa and less than 100KDa target protein, transferring film condition be semidry method transferring film, 3mA,
3.5min；
It is less than or equal to 40KDa target protein for molecular weight, transferring film condition is semidry method transferring film, 3mA, 2.5min.
8. the assay method of the fribrillin serine phosphorylation level as claimed in claim 5 for avoiding signal from disturbing,
Characterized in that, in the serine phosphorus for the fribrillin sample that the phosphorylation is detected by protein immunoblotting method
During acidifying level, before being mixed with the first antibody, rule along albumen marker pencil levels and extend to albumen
Vacant band between marker and fribrillin, and the band is vertically cut off, by remaining fribrillin part with
The first antibody mixing is incubated.
9. the assay method of the fribrillin serine phosphorylation level as claimed in claim 8 for avoiding signal from disturbing,
Characterized in that, in the serine phosphorus for the fribrillin sample that the phosphorylation is detected by protein immunoblotting method
During acidifying level,
If detecting the non-myoglobulin heavy chain of target, actin, before being mixed with first antibody, cut along albumen marker levels
Striping middle-molecular-weihydroxyethyl is more than 220KDa bands, 40-45KDa strip portions, and remainder corner cut is marked, mixed with first antibody
Close；
If it is myoglobulin heavy chain and actin to detect target, before being mixed with first antibody, cut along albumen marker levels
Part and 34-55KDa strip portions, remainder corner cut is marked below striping middle-molecular-weihydroxyethyl 180KDa bands.
10. the assay method of the fribrillin serine phosphorylation level as claimed in claim 1 for avoiding signal from disturbing,
Characterized in that, the fribrillin sample is derived from sheep.
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