CN107219369B - Avoid the measuring method of the fribrillin serine phosphorylation level of signal interference - Google Patents
Avoid the measuring method of the fribrillin serine phosphorylation level of signal interference Download PDFInfo
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Abstract
The measuring method of the fribrillin serine phosphorylation level provided by the invention for avoiding signal interference, detect that different fribrillin serine phosphorylations are horizontal 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 the method for fribrillin serine phosphorylation level in the efficient measurement postmortem muscle of one kind.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 field of biotechnology more particularly to a kind of fribrillin serines for avoiding signal interference
The measuring method of phosphorylation level.
Background technique
Protein phosphorylation modification is the important channel of regulation protein structure and function, at present about protein phosphorylation
Horizontal method is concentrated mainly on the whole phosphorylation level of measurement.Phosphorylation modification is likely to occur in serine, threonine, junket ammonia
On acid, wherein the phosphorylation modification of serine is that quantity is most, and the Biochemical changes of fribrillin phosphorylation are carried out
Research, and based on fribrillin phosphorylation to the research of animal muscle Physiology and biochemistry be in scientific research it is essential
's.Although also there is the specific antibody for certain amino acid in the prior art, fribrillin is many kinds of, all kinds of
Between content difference it is huge, although mass spectrum can detecte the phosphorylation level and phosphorylation site of multiple protein, single
Test sample is few, and price is high, and data analysis is complicated, and there is presently no horizontal for certain fribrillin serine phosphorylation
Immune blotting detection method.Therefore, one kind can polyprotein identification, detect quick, low-cost measurement fribrillin silk
The method of propylhomoserin phosphorylation level needs.
Summary of the invention
It is excellent it is an object of the invention to solve at least the above problems and/or defect, and provide at least to will be described later
Point.
It is a still further object of the present invention to provide a kind of fribrillin serine phosphorylation water for avoiding signal interference
Flat measuring method is distinguished different fribrillins by different fribrillin contents, molecular weight, signal response
Carry out immunoblotting.
A further object of the present invention is to provide a kind of fribrillin serine phosphorylation water for avoiding signal interference
Flat measuring method avoids serine phosphorylation signal between various fribrillins from interfering with each other, by utilizing Diagnosis of Sghistosomiasis
Mark realizes the measurement to different fribrillin phosphorylation levels, and a certain kind cannot accurately be detected by solving in existing method
The problem of fribrillin, cumbersome, somewhat expensive.
The present invention provides a kind of measuring method of fribrillin serine phosphorylation level for avoiding signal interference, packet
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 runs to apart from glue substrate 0.5mm to fribrillin sample bromophenol blue label and stops electrophoresis；
It is horizontal that serine phosphorylation in fribrillin sample is detected by protein immunoblotting method.
Preferably, the measuring method of the fribrillin serine phosphorylation level for avoiding signal interference
In, a blank swimming lane is stayed between two swimming lanes of albumen marker and fribrillin.
Preferably, the measuring method of the fribrillin serine phosphorylation level for avoiding signal interference
In,
It is more than or equal to the target protein of 100KDa for molecular weight, selected resolving gel concentration is 7.5%；
40KDa is higher than for molecular weight and is less than the target protein of 100KDa, selected resolving gel concentration is 10%；
It is less than or equal to the target protein of 40KDa for molecular weight, selected resolving gel concentration is 12%；
Preferably, the measuring method of the fribrillin serine phosphorylation level for avoiding signal interference
In,
Albumen marker applied sample amount is 2 μ L；
Myoglobulin heavy chain higher for content in fribrillin, actin applied sample amount are 2.5 μ g, for it
His fribrillin applied sample amount is 10 μ g.
Preferably, the measuring method of the fribrillin serine phosphorylation level for avoiding signal interference
In, it is detected in the fribrillin serine phosphorylation horizontal process by protein immunoblotting method, 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 measuring method of the fribrillin serine phosphorylation level for avoiding signal interference
In, it is detected in the fribrillin serine phosphorylation horizontal process by protein immunoblotting method, with described the
The secondary antibody that one antibody combines uses sheep anti-mouse igg, and the concentration of the secondary antibody is 0.05-0.2 μ g/mL.
Preferably, the measuring method of the fribrillin serine phosphorylation level for avoiding signal interference
In, it is horizontal in the serine phosphorylation for detecting the fribrillin sample of the phosphorylation by protein immunoblotting method
In the process,
It is more than or equal to the target protein of 100KDa for molecular weight, transferring film condition is semidry method transferring film, 3mA, 4.5min；
For molecular weight be higher than 40KDa and be less than 100KDa target protein, transferring film condition be semidry method transferring film, 3mA,
3.5min；
It is less than or equal to the target protein of 40KDa for molecular weight, transferring film condition is semidry method transferring film, 3mA, 2.5min.
Preferably, the measuring method of the fribrillin serine phosphorylation level for avoiding signal interference
In, it is horizontal in the serine phosphorylation for detecting the fribrillin sample of the phosphorylation by protein immunoblotting method
In the process, before being mixed with the first antibody, cross along albumen marker pencil level 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 for.
Preferably, the measuring method of the fribrillin serine phosphorylation level for avoiding signal interference
In, it is horizontal in the serine phosphorylation for detecting the fribrillin sample of the phosphorylation by protein immunoblotting method
In the process,
If the non-myoglobulin heavy chain of target, actin are detected, before mixing with first antibody, along albumen marker water
Straight snips striping middle-molecular-weihydroxyethyl is greater than 220KDa band, 40-45KDa strip portion, remainder corner cut is marked, with first antibody
Mixing；
If detecting target is myoglobulin heavy chain and actin, before mixing with first antibody, along albumen marker water
Part and 34-55KDa strip portion, remainder corner cut is marked below straight snips striping middle-molecular-weihydroxyethyl 180KDa band.
Preferably, the measuring method of the fribrillin serine phosphorylation level for avoiding signal interference
In, the measuring method of the fribrillin serine phosphorylation level for avoiding signal interference, which is characterized in that described
Fribrillin sample is derived from sheep.
The present invention is include at least the following beneficial effects:
The measuring method of the fribrillin serine phosphorylation level provided by the invention for avoiding signal interference, passes through
Protein immunoblotting technology can equally detect that different fribrillin serine phosphorylations are horizontal in postmortem muscle,
Required muscle samples amount is few, and operating process is relatively simple, reproducible, and detection gained band is clear, and it is one that experiment price is low
The method that kind efficiently measures fribrillin serine phosphorylation level in postmortem muscle.It can be to ovine skeletal muscle using this method
The Biochemical changes of middle serine phosphorylation fribrillin are researched and analysed, and it is raw also to may extend to other animal muscle physiology
The research of change.
Further advantage, target and feature of the invention will be partially reflected by the following instructions, and part will also be by this
The research and practice of utility model and be understood by the person skilled in the art.
Detailed description of the invention
Fig. 1 is the measuring method of the fribrillin serine phosphorylation level provided by the invention for avoiding signal interference
Embodiment 1 in various concentration gel myosin image after different time transferring film；
Fig. 2 is the measuring method of the fribrillin serine phosphorylation level provided by the invention for avoiding signal interference
Embodiment 1 in various concentration gel desmin image after different time transferring film；
Fig. 3 is the measuring method of the fribrillin serine phosphorylation level provided by the invention for avoiding signal interference
Embodiment 1 in various concentration gel actin image after different time transferring film；
Fig. 4 is the measuring method of the fribrillin serine phosphorylation level provided by the invention for avoiding signal interference
Embodiment 1 in various concentration gel troponin T image after different time transferring film；
Fig. 5 is the measuring method of the fribrillin serine phosphorylation level provided by the invention for avoiding signal interference
Embodiment 2 in albumen marker to exposure signal interference figure；
Fig. 6 is the measuring method of the fribrillin serine phosphorylation level provided by the invention for avoiding signal interference
Embodiment 4 in myoglobulin heavy chain serine phosphorylation exposure image figure；
Fig. 7 is the measuring method of the fribrillin serine phosphorylation level provided by the invention for avoiding signal interference
Embodiment 4 in actin serine phosphorylation exposure image figure；
Fig. 8 is the measuring method of the fribrillin serine phosphorylation level provided by the invention for avoiding signal interference
Embodiment 5 in fribrillin (except myoglobulin heavy chain and actin) serine phosphorylation exposure image figure.
Specific embodiment
The following describes the utility model in further detail with reference to the accompanying drawings, to enable those skilled in the art referring to explanation
Book text can be implemented accordingly.
It should be appreciated that such as " having ", "comprising" and " comprising " term used herein do not allot one or more
The presence or addition of a other elements or combinations thereof.
The present invention provides a kind of measuring method of fribrillin serine phosphorylation level for avoiding signal interference, packet
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 runs to apart from glue substrate 0.5mm to fribrillin sample bromophenol blue label and stops electrophoresis；
It is horizontal that serine phosphorylation in fribrillin sample is detected by protein immunoblotting method.
In the measuring method of the fribrillin serine phosphorylation level for avoiding signal interference, albumen
A blank swimming lane is stayed between two swimming lanes of marker and fribrillin.
In the measuring method of the fribrillin serine phosphorylation level for avoiding signal interference,
It is more than or equal to the target protein of 100KDa for molecular weight, selected resolving gel concentration is 7.5%；
40KDa is higher than for molecular weight and is less than the target protein of 100KDa, selected resolving gel concentration is 10%；
It is less than or equal to the target protein of 40KDa for molecular weight, selected resolving gel concentration is 12%；
In the measuring method of the fribrillin serine phosphorylation level for avoiding signal interference,
Albumen marker applied sample amount is 2 μ L；
Myoglobulin heavy chain higher for content in fribrillin, actin applied sample amount are 2.5 μ g, for it
His fribrillin applied sample amount is 10 μ g.
In the measuring method of the fribrillin serine phosphorylation level for avoiding signal interference, pass through albumen
Matter western blotting method detects 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 measuring method of the fribrillin serine phosphorylation level for avoiding signal interference, pass through albumen
Matter western blotting method detects in the fribrillin serine phosphorylation horizontal process, in conjunction 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 measuring method of the fribrillin serine phosphorylation level for avoiding signal interference, passing through egg
White matter western blotting method detects in the serine phosphorylation horizontal process of the fribrillin sample of the phosphorylation,
It is more than or equal to the target protein of 100KDa for molecular weight, transferring film condition is semidry method transferring film, 3mA, 4.5min；
For molecular weight be higher than 40KDa and be less than 100KDa target protein, transferring film condition be semidry method transferring film, 3mA,
3.5min；
It is less than or equal to the target protein of 40KDa for molecular weight, transferring film condition is semidry method transferring film, 3mA, 2.5min.
In the measuring method of the fribrillin serine phosphorylation level for avoiding signal interference, passing through egg
White matter western blotting method detects in the serine phosphorylation horizontal process of the fribrillin sample of the phosphorylation, with institute
Before stating first antibody mixing, crosses along albumen marker pencil level 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 measuring method of the fribrillin serine phosphorylation level for avoiding signal interference, passing through egg
White matter western blotting method detects in the serine phosphorylation horizontal process of the fribrillin sample of the phosphorylation,
If the non-myoglobulin heavy chain of target, actin are detected, before mixing with first antibody, along albumen marker water
Straight snips striping middle-molecular-weihydroxyethyl is greater than 220KDa band, 40-45KDa strip portion, remainder corner cut is marked, with first antibody
Mixing；
If detecting target is myoglobulin heavy chain and actin, before mixing with first antibody, along albumen marker water
Part and 34-55KDa strip portion, remainder corner cut is marked below straight snips striping middle-molecular-weihydroxyethyl 180KDa band.
In the measuring method of the fribrillin serine phosphorylation level for avoiding signal interference, described is kept away
Exempt from the measuring method of the fribrillin serine phosphorylation level of signal interference, which is characterized 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, glycerol 2mL, 0.5M Tris-HCl pH6.8 2mL, 1M
DTT 2.0mL, bromophenol blue 0.01g:
7.5%, 10%, 12% separation gel and 4% concentration glue: Bio-Rad#1610181, #1610183, #
1610185；
TBS buffer: the HCl tune pH to 7.5 of Tris 1.211g, NaCl 8.76g, 1mol/L；
Include in confining liquid: 25 μ l/L, BSA 1.5g/L of TBS 50ml/L, Tween20:
TBST1: every 500mLTBS adds 0.5mL Tween20；
TBST2:Tris 3.0275g, NaCl 4.38g, Tween20 0.5mL, HCl tune pH to 7.5, is settled to 500mL；
Protein standards: thermo#26616；
Kit used in ECL development process are as follows: Bio-Rad#1705061；
The kit that the protein concentration that sample is extracted in the measurement of BCA method uses is Thermo#23225；
Following embodiments are all made of the unidirectional electrophoresis system of Bio-Rad, Bio-Rad semidry method transferring film instrument and Bio-Rad gel
Imaging system.
Embodiment 1
A) the sheep longissimus dorsi muscle fribrillin sample of -80 DEG C of preservations in part is taken, BCA method measures 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 amount is 2 μ L, flesh ball higher for 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 for being equal to 100KDa, using 7.5% separation gel, 40KDa is higher than for molecular weight and is less than the mesh of 100KDa
Albumen is marked, using 10% separation gel, the target protein of 40KDa is less than or equal to for molecular weight, using 12% separation gel；Electricity
Voltage of swimming uses constant pressure 200V, runs to sample bromophenol blue label to apart from 0.5 mm of glue substrate and stops electrophoresis；
C) protein immunoblotting (IB): unloading offset plate after electrophoresis, gel is put into transferring film buffer and is balanced；
PVDF film activates 15s with pure methanol, is put into togerther in transferring film buffer and balances 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 the target of 100KDa for molecular weight
Albumen, transferring film condition are semidry method transferring film, and 3mA, 4.5min are higher than 40KDa for molecular weight and are less than the target egg of 100KDa
White, transferring film condition is semidry method transferring film, and 3mA, 3.5min are less than or equal to molecular weight the target protein of 40KDa, transferring film condition
For semidry method transferring film, 3mA, 2.5min；After transferring film, PVDF film is washed three times with TBS buffer, each 3min；
Pvdf membrane is put into the hybridization bag cut, 5ml confining liquid is added, at room temperature the concussion closing 2h on shaking table；
First antibody abbreviation primary antibody, primary antibody are incubated for: the phosphorylation serine list of Sigma company being added in 5ml confining liquid
Clonal antibody (article No.: P3430), concentration are 70 μ g/ml；Hybridization bag is replaced, by 4 DEG C of pvdf membrane and prepared primary antibody solution
It is incubated overnight；
Secondary antibody abbreviation secondary antibody, secondary antibody are incubated for: washing pvdf membrane three times with TBST1 buffer, each 20min；In 5ml
The anti-mouse IgG (article No. A9044) of Sigma company is added in confining liquid, concentration is 0.1 μ g/ml, molten as secondary antibody
Liquid and the concussion of pvdf membrane room temperature are incubated for 2h；
Pvdf membrane is washed three times with TBST2 buffer, and each 10min utilizes the ECL (article No. of Bio-rid company later
For) colour developing.
Embodiment 2
A) the sheep longissimus dorsi muscle fribrillin sample of -80 DEG C of preservations in part is taken, BCA method measures 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 amount is 2 μ L, flesh ball higher for 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 for being equal to 100KDa, using 7.5% separation gel, 40KDa is higher than for molecular weight and is less than the mesh of 100KDa
Albumen is marked, using 10% separation gel, the target protein of 40KDa is less than or equal to for molecular weight, using 12% separation gel；Electricity
Voltage of swimming uses constant pressure 200V, runs to sample bromophenol blue label to apart from 0.5 mm of glue substrate and stops electrophoresis；
C) protein immunoblotting (IB): unloading offset plate after electrophoresis, gel is put into transferring film buffer and is balanced；
PVDF film activates 15s with pure methanol, is put into togerther in transferring film buffer and balances 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 the target of 100KDa for molecular weight
Albumen, transferring film condition are semidry method transferring film, and 3mA, 4.5min are higher than 40KDa for molecular weight and are less than the target egg of 100KDa
White, transferring film condition is semidry method transferring film, and 3mA, 3.5min are less than or equal to molecular weight the target protein of 40KDa, transferring film condition
For semidry method transferring film, 3mA, 2.5min；After transferring film, PVDF film is washed three times with TBS buffer, each 3min；
Pvdf membrane is put into the hybridization bag cut, 5ml confining liquid is added, at room temperature the concussion closing 2h on shaking table；
Albumen marker is cut from pvdf membrane；
First antibody abbreviation primary antibody, primary antibody are incubated for: the phosphorylation serine list of Sigma company being added in 5ml confining liquid
Clonal antibody (article No.: P3430), concentration are 70 μ g/ml；Hybridization bag is replaced, pvdf membrane and the preparation of albumen marker will be cut
Good 4 DEG C of primary antibody solution are incubated overnight；
Secondary antibody abbreviation secondary antibody, secondary antibody are incubated for: washing pvdf membrane three times with TBST1 buffer, each 20min；In 5ml
The anti-mouse IgG (article No. A9044) of Sigma company is added in confining liquid, concentration is 0.1 μ g/ml, molten as secondary antibody
Liquid and the concussion of pvdf membrane room temperature are incubated for 2h；
Pvdf membrane is washed three times with TBST2 buffer, and each 10min utilizes the ECL (article No. of Bio-rid company later
For) colour developing.
Embodiment 3
A) the sheep longissimus dorsi muscle fribrillin sample of -80 DEG C of preservations in part is taken, BCA method measures 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 amount is 2 μ L, flesh ball higher for 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 for being equal to 100KDa, using 7.5% separation gel, 40KDa is higher than for molecular weight and is less than the mesh of 100KDa
Albumen is marked, using 10% separation gel, the target protein of 40KDa is less than or equal to for molecular weight, using 12% separation gel；Egg
Loading, electrophoretic voltage do not use constant pressure 200V to the one blank swimming lane of interval of white marker and sample, mark and run to sample bromophenol blue
Stop electrophoresis to apart from glue substrate 0.5mm；
C) protein immunoblotting (IB): unloading offset plate after electrophoresis, gel is put into transferring film buffer and is balanced；
PVDF film activates 15s with pure methanol, is put into togerther in transferring film buffer and balances 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 the target of 100KDa for molecular weight
Albumen, transferring film condition are semidry method transferring film, and 3mA, 4.5min are higher than 40KDa for molecular weight and are less than the target egg of 100KDa
White, transferring film condition is semidry method transferring film, and 3mA, 3.5min are less than or equal to molecular weight the target protein of 40KDa, transferring film condition
For semidry method transferring film, 3mA, 2.5min；After transferring film, PVDF film is washed three times with TBS buffer, each 3min；
Pvdf membrane is put into the hybridization bag cut, 5ml confining liquid is added, at room temperature the concussion closing 2h on shaking table；
It is crossed along albumen marker level with pencil, extends to item vacant between albumen marker and fribrillin
Band cuts albumen marker from pvdf membrane among blank band between albumen marker and sample；
First antibody abbreviation primary antibody, primary antibody are incubated for: the phosphorylation serine list of Sigma company being added in 5ml confining liquid
Clonal antibody (article No.: P3430), concentration are 70 μ g/ml；Hybridization bag is replaced, pvdf membrane and the preparation of albumen marker will be cut
Good 4 DEG C of primary antibody solution are incubated overnight；
Secondary antibody abbreviation secondary antibody, secondary antibody are incubated for: washing pvdf membrane three times with TBST1 buffer, each 20min；In 5ml
The anti-mouse IgG (article No. A9044) of Sigma company is added in confining liquid, concentration is 0.1 μ g/ml, molten as secondary antibody
Liquid and the concussion of pvdf membrane room temperature are incubated for 2h；
Pvdf membrane is washed three times with TBST2 buffer, and each 10min utilizes the ECL (article No. of Bio-rid company later
For) colour developing.
Embodiment 4
A) the sheep longissimus dorsi muscle fribrillin sample of -80 DEG C of preservations in part is taken, BCA method measures 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 amount is 2 μ L, flesh ball higher for 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 for being equal to 100KDa, using 7.5% separation gel, 40KDa is higher than for molecular weight and is less than the mesh of 100KDa
Albumen is marked, using 10% separation gel, the target protein of 40KDa is less than or equal to for molecular weight, using 12% separation gel；Egg
Loading, electrophoretic voltage do not use constant pressure 200V to the one blank swimming lane of interval of white marker and sample, mark and run to sample bromophenol blue
Stop electrophoresis to apart from glue substrate 0.5mm；
C) protein immunoblotting (IB): unloading offset plate after electrophoresis, gel is put into transferring film buffer and is balanced；
PVDF film activates 15s with pure methanol, is put into togerther in transferring film buffer and balances 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 the target of 100KDa for molecular weight
Albumen, transferring film condition are semidry method transferring film, and 3mA, 4.5min are higher than 40KDa for molecular weight and are less than the target egg of 100KDa
White, transferring film condition is semidry method transferring film, and 3mA, 3.5min are less than or equal to molecular weight the target protein of 40KDa, transferring film condition
For semidry method transferring film, 3mA, 2.5min；After transferring film, PVDF film is washed three times with TBS buffer, each 3min；
Pvdf membrane is put into the hybridization bag cut, 5ml confining liquid is added, at room temperature the concussion closing 2h on shaking table；
It is crossed along albumen marker level with pencil, extends to item vacant between albumen marker and fribrillin
Band cuts albumen marker from pvdf membrane among blank band between albumen marker and sample；
It is horizontal along albumen marker before being mixed with first antibody if detection target is actomyosin, actin
Film middle-molecular-weihydroxyethyl 180KDa band or less part and 34-55KDa strip portion are cut off, remainder corner cut is marked；
First antibody abbreviation primary antibody, primary antibody are incubated for: the phosphorylation serine list of Sigma company being added in 5ml confining liquid
Clonal antibody (article No.: P3430), concentration are 70 μ g/ml；Hybridization bag is replaced, pvdf membrane and the preparation of albumen marker will be cut
Good 4 DEG C of primary antibody solution are incubated overnight；
Secondary antibody abbreviation secondary antibody, secondary antibody are incubated for: washing pvdf membrane three times with TBST1 buffer, each 20min；In 5ml
The anti-mouse IgG (article No. A9044) of Sigma company is added in confining liquid, concentration is 0.1 μ g/ml, molten as secondary antibody
Liquid and the concussion of pvdf membrane room temperature are incubated for 2h；
Pvdf membrane is washed three times with TBST2 buffer, and each 10min utilizes the ECL (article No. of Bio-rid company later
For) colour developing.
Embodiment 5
A) the sheep longissimus dorsi muscle fribrillin sample of -80 DEG C of preservations in part is taken, BCA method measures 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 amount is 2 μ L, flesh ball higher for 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 for being equal to 100KDa, using 7.5% separation gel, 40KDa is higher than for molecular weight and is less than the mesh of 100KDa
Albumen is marked, using 10% separation gel, the target protein of 40KDa is less than or equal to for molecular weight, using 12% separation gel；Egg
Loading, electrophoretic voltage do not use constant pressure 200V to the one blank swimming lane of interval of white marker and sample, mark and run to sample bromophenol blue
Stop electrophoresis to apart from glue substrate 0.5mm；
C) protein immunoblotting (IB): unloading offset plate after electrophoresis, gel is put into transferring film buffer and is balanced；
PVDF film activates 15s with pure methanol, is put into togerther in transferring film buffer and balances 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 the target of 100KDa for molecular weight
Albumen, transferring film condition are semidry method transferring film, and 3mA, 4.5min are higher than 40KDa for molecular weight and are less than the target egg of 100KDa
White, transferring film condition is semidry method transferring film, and 3mA, 3.5min are less than or equal to molecular weight the target protein of 40KDa, transferring film condition
For semidry method transferring film, 3mA, 2.5min；After transferring film, PVDF film is washed three times with TBS buffer, each 3min；
Pvdf membrane is put into the hybridization bag cut, 5ml confining liquid is added, at room temperature the concussion closing 2h on shaking table；
It is crossed along albumen marker level with pencil, extends to item vacant between albumen marker and fribrillin
Band cuts albumen marker from pvdf membrane among blank band between albumen marker and sample；
It is horizontal along albumen marker before being mixed with first antibody if detecting the non-actomyosin of target, actin
Film middle-molecular-weihydroxyethyl is cut off greater than 220KDa band, 40-45KDa strip portion, remainder corner cut is marked；
First antibody abbreviation primary antibody, primary antibody are incubated for: the phosphorylation serine list of Sigma company being added in 5ml confining liquid
Clonal antibody (article No.: P3430), concentration are 70 μ g/ml；Hybridization bag is replaced, pvdf membrane and the preparation of albumen marker will be cut
Good 4 DEG C of primary antibody solution are incubated overnight；
Secondary antibody abbreviation secondary antibody, secondary antibody are incubated for: washing pvdf membrane three times with TBST1 buffer, each 20min；In 5ml
The anti-mouse IgG (article No. A9044) of Sigma company is added in confining liquid, concentration is 0.1 μ g/ml, molten as secondary antibody
Liquid and the concussion of pvdf membrane room temperature are incubated for 2h；
Pvdf membrane is washed three times with TBST2 buffer, and each 10min utilizes the ECL (article No. of Bio-rid company later
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, and target is other albumen, and applied sample amount is 10 μ g, big for molecular weight
In be equal to 100KDa target protein, transferring film condition be semidry method transferring film, 3mA, 4.5min, for molecular weight be higher than 40KDa and
Target protein less than 100KDa, transferring film condition are 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 220kDa, between flesh
Linear protein molecular weight is about 55kDa, and actin molecular weight is about 43 kDa, and troponin T molecular weight is about 37kDa.Using
Above-mentioned condition, it is detected with corresponding antibody test myoglobulin heavy chain, desmin, actin, troponin T
Band it is clear, and after transferring film on pvdf membrane without target protein remain.
Fig. 5 is that embodiment 2 is resulting as a result, as shown in figure 5, using automatic exposure, can only observe clearly albumen
The band of marker illustrates marker there is also serine phosphorylation phenomenon, after overexposure, it may be observed that fribrillin
In the serine phosphorylations of some albumen modify phenomenon, but albumen marker generates stronger signal interference to neighbouring lane, cuts
Overexposure is carried out after falling marker, the serine phosphorylation signal of some albumen is stronger in fribrillin, but with
Marker adjacent swimming lane still has signal interference phenomenon.
Fig. 6 and Fig. 7 is that embodiment 4 is resulting as a result, simultaneously using the method for embodiment 3, as shown in figure 3, flesh ball egg
Bai Chonglian and actin are the highest two kinds of albumen of content in fribrillin, by molecular weight ranges where two albumen
Part is cut from pvdf membrane before primary antibody is incubated for, available clearly serine phosphorylation band, and by albumen marker
With sample every a swimming lane loading, the signal interference of marker is successfully eliminated.
Fig. 8 is that embodiment 5 is resulting as a result, simultaneously using the method for embodiment 3, as shown in figure 8, by myosin weight
After chain and actin are cut from pvdf membrane, other albumen that can successfully avoid both albumen relatively low to content
Signal interference can detect a plurality of serine phosphorylation band；And by albumen marker and sample every a swimming lane loading, success
Eliminate the signal interference of marker；It simultaneously before cutting albumen marker, is crossed, is extended with pencil along albumen marker level
It, can be dry excluding albumen marker with labelled protein molecular weight to band vacant between albumen marker and fribrillin
Also the effect of albumen marker has been played while disturbing.
Treatment scale described herein is for simplifying explanation of the invention.To Phosphorylation and albumen of the invention
The application of the methods of matter immunoprecipitation and immunoblotting, modifications and variations are apparent to one skilled in the art
's.
As described above, according to the present invention, detect fribrillin serine in sheep longissimus dorsi muscle and achieve success, and,
It is tested repeatedly, the method for the present invention experimental repeatability is good, can be fribrillin silk in the animals skeletal muscles such as sheep as a result reliably
The detection research of propylhomoserin phosphorylation provides technical example.
Although the embodiments of the present invention have been disclosed as above, but its is not only in the description and the implementation listed
With it can be fully applied to various fields suitable for the present invention, for those skilled in the art, can be easily
Realize other modification, therefore without departing from the general concept defined in the claims and the equivalent scope, the present invention is simultaneously unlimited
In specific details and legend shown and described herein.
Claims (8)
1. avoiding the measuring method of the fribrillin serine phosphorylation level of signal interference, which is characterized 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 runs to apart from glue substrate 0.5mm to fribrillin sample bromophenol blue label and stops electrophoresis；
It is horizontal that serine phosphorylation in fribrillin sample is detected by protein immunoblotting method；
Wherein,
A blank swimming lane is stayed between two swimming lanes of albumen marker and fribrillin；
Before being mixed with first antibody, crosses along albumen marker pencil level and extend to albumen marker and muscle fibril
Vacant band between albumen, and the band is vertically cut off, remaining fribrillin part is mixed with the first antibody
It is incubated for.
2. the measuring method of the fribrillin serine phosphorylation level of signal interference is avoided as described in claim 1,
It is characterized in that,
It is more than or equal to the target protein of 100KDa for molecular weight, selected resolving gel concentration is 7.5%；
40KDa is higher than for molecular weight and is less than the target protein of 100KDa, selected resolving gel concentration is 10%；
It is less than or equal to the target protein of 40KDa for molecular weight, selected resolving gel concentration is 12%.
3. the measuring method of the fribrillin serine phosphorylation level of signal interference is avoided as described in claim 1,
It is characterized in that,
Albumen marker applied sample amount is 2 μ L；
Myoglobulin heavy chain higher for content in fribrillin, actin applied sample amount are 2.5 μ g, for other fleshes
Fibrillin applied sample amount is 10 μ g.
4. the measuring method of the fribrillin serine phosphorylation level of signal interference is avoided as described in claim 1,
It is characterized in that, detected in the fribrillin serine phosphorylation horizontal process by protein immunoblotting method,
The first antibody reacted with the fribrillin uses serine phosphorylation monoclonal antibody, the concentration of the first antibody
For 60-80 μ g/ml.
5. the measuring method of the fribrillin serine phosphorylation level of signal interference is avoided as claimed in claim 4,
It is characterized in that, detected in the fribrillin serine phosphorylation horizontal process by protein immunoblotting method,
Secondary antibody in conjunction with the first antibody uses sheep anti-mouse igg, and the concentration of the secondary antibody is 0.05-0.2 μ g/mL.
6. the measuring method of the fribrillin serine phosphorylation level of signal interference is avoided as claimed in claim 2,
It is characterized in that, in the serine phosphorus for the fribrillin sample for detecting the phosphorylation by protein immunoblotting method
During acidification is horizontal,
It is more than or equal to the target protein of 100KDa for molecular weight, transferring film condition is semidry method transferring film, 3mA, 4.5min；
For molecular weight be higher than 40KDa and be less than 100KDa target protein, transferring film condition be semidry method transferring film, 3mA,
3.5min；
It is less than or equal to the target protein of 40KDa for molecular weight, transferring film condition is semidry method transferring film, 3mA, 2.5min.
7. the measuring method of the fribrillin serine phosphorylation level of signal interference is avoided as claimed in claim 6,
It is characterized in that, in the serine phosphorus for the fribrillin sample for detecting the phosphorylation by protein immunoblotting method
During acidification is horizontal,
If the non-myoglobulin heavy chain of detection target, actin are cut before mixing with first antibody along albumen marker level
Striping middle-molecular-weihydroxyethyl is greater than 220KDa band, 40-45KDa strip portion, and remainder corner cut is marked, mixed with first antibody
It closes；
If detecting target is myoglobulin heavy chain and actin, before being mixed with first antibody, cut along albumen marker level
Part and 34-55KDa strip portion, remainder corner cut is marked below striping middle-molecular-weihydroxyethyl 180KDa band.
8. the measuring method of the fribrillin serine phosphorylation level of signal interference is avoided as described in claim 1,
It is characterized in that, the fribrillin sample is derived from sheep.
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