CN105334329B - The assay method of calpain phosphorylation level - Google Patents
The assay method of calpain phosphorylation level Download PDFInfo
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- CN105334329B CN105334329B CN201510919423.8A CN201510919423A CN105334329B CN 105334329 B CN105334329 B CN 105334329B CN 201510919423 A CN201510919423 A CN 201510919423A CN 105334329 B CN105334329 B CN 105334329B
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Abstract
The invention discloses a kind of assay method of calpain phosphorylation level, including：Step 1: the protein concentration of the protein sample containing calpain is adjusted into 5.83~12.5 μ g/ μ l, the mass ratio of the overall reaction amount of the albumen added thereto afterwards in phospho-AB, phospho-AB and the protein sample containing calpain is 1:1750~2500, then the two is incubated in concussion at 4 DEG C, to form immune complex, then the calpain sample for obtaining phosphorylation is enriched with by Western Immuno intermediate processing；And, Step 2: passing through the phosphorylation level of the calpain sample of phosphorylation obtained in protein immunoblotting method detecting step one.The present invention can detect the calpain of phosphorylation by Western Immuno precipitation and immunoblot assay in postmortem muscle, required muscle samples amount is few, and operating process is simple, reproducible, detection gained band is clear, and potential injury of the test method to human body is small.
Description
Technical field
The invention belongs to biological technical field, it is related to a kind of assay method of calpain phosphorylation level, the technology can
Directly apply to sheep and the biochemical research of other animal muscles.
Background technology
The phosphorus of calpain in organism is detected typically by isotope-labelling method in the life science such as medical science field
Acidifying level, but the method for isotope marks detection is cumbersome and very big to the potential injury of human body.But, for phosphorylation calcium
The Biochemical changes of protease are researched and analysed, and for the research of other animal muscle Physiology and biochemistries, calpain
Phosphorylation is essential in scientific research.Determined more for protease phosphorylation level although also having in the prior art
The mode of safety, but be due to that calpain is more sensitive, easily it is affected by the external environment, causes activity reduction or even inactivation etc.
Characteristic.So, there is presently no the method detected specifically designed for calpain phosphorylation level.Therefore, it is a kind of it is safe to the human body,
The method that detection rapidly and accurately determines the phosphorylation of calpain 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 extracting method of calpain crude samples, pass through the tissue of the present invention
The content of calpain is higher, active higher in the protein sample that lysate is extracted, and solves in phosphorylation level continuous mode
The problem of middle easy partial inactivation of calpain or all inactivation.
A further object of the present invention is to provide a kind of assay method of calpain phosphorylation level, and the present invention passes through profit
The measure to calpain phosphorylation level is realized with protein immunization precipitation and protein immunization technology, is solved in existing method
The problems such as harm to the human body is larger and operating process is cumbersome when determining phosphorylation level.
The technical scheme that the present invention is provided is：
A kind of assay method of calpain phosphorylation level, including：
Step 1: the protein concentration of the protein sample containing calpain is adjusted into 5.83~12.5 μ g/ μ l, it is backward its
Middle addition phospho-AB, the overall reaction amount of the phospho-AB and the albumen in the protein sample containing calpain
Mass ratio be 1:1750~2500, then the protein sample containing calpain is incubated with phospho-AB in concussion at 4 DEG C
Educate, to form immune complex, then the calpain sample for obtaining phosphorylation is enriched with by Western Immuno intermediate processing；With
And,
Step 2: detecting the calcium albumen of the phosphorylation obtained in the step one by protein immunoblotting method
The phosphorylation level of enzyme sample.
Preferably, in the assay method of described calpain phosphorylation level, in the step one, protein is passed through
The specific steps for the calpain sample that immunoprecipitation method enrichment obtains the phosphorylation include：
1.1) Protein A/G agarose resins slurries are taken and make its absorption on centrifugal column, wherein, the Protein
The volume ratio of A/G agarose resins slurries and the calcium protein sample is 2~3:The institute added in 40~60, and each centrifugal column
The volume for stating Protein A/G agarose resin slurries is 20-30 μ l；
1.2) by the immune complex add by step 1.1) handle after be adsorbed with the Protein A/G agar
In the centrifugal column of glucoresin, and 1h is incubated in 4 DEG C of isothermal vibrations, centrifuges, then washed using IP lavation buffer solutions afterwards
The Protein A/G agarose resins three times, then wash with IP condition buffer solutions the Protein A/G agarose resins one
Secondary, what is retained on the last centrifugal column is the ternary of calpain, phospho-AB and Protein A/G agarose resins
Compound；With,
1.3) to step 1.2) in retention have reproducibility loading buffer added in the centrifugal column of the ternary complex
Liquid, and in being incubated 5min at 100 DEG C, 10000 × g centrifuges 1min at room temperature after cooling, and the liquid that flows through of gained is enrichment
The phosphorylation calpain sample.
Preferably, in the assay method of described calpain phosphorylation level, in the step 2, albumen is being passed through
Matter western blotting method is detected during the phosphorylation level of the calpain sample of the phosphorylation, anti-with the calpain
The first antibody answered uses μ-calpain monoclonal antibodies, and the concentration of the first antibody is 1.4-2 μ g/ml.
Preferably, in the assay method of described calpain phosphorylation level, in the step 2, albumen is being passed through
Matter western blotting method is detected during the phosphorylation level of the calpain sample of the phosphorylation, with the first antibody knot
The secondary antibody of conjunction uses anti-mouse IgG, and the concentration of the secondary antibody is 0.1-0.3 μ g/ml.
Preferably, in the assay method of described calpain phosphorylation level, also include before the step one
The method for extracting the protein sample containing calpain：
Animal muscle sample is gathered, and according to mass volume ratio 1:The animal muscle sample is added Tissue lysates by 6
Or in sarcoplasm extract, by the animal muscle sample and the Tissue lysates in being homogenized on ice to the animal muscle sample
Product and the Tissue lysates form homogeneous homogenate, then produce the homogenate in supernatant is collected by centrifugation at 4 DEG C
To the protein sample containing calpain；And all steps are in operation on ice or ice bath operation；
Wherein, the Tissue lysates are 25mM bicine and 150mM sodium chloride, the tissue comprising concentration
The pH of lysate is 7.6；
The sarcoplasm extract includes sulfydryl second of the concentration for 100mM Tris base, 10mM EDTA and 0.05%
Alcohol, the pH of the sarcoplasm extract is 8.3.
Preferably, in the assay method of described calpain phosphorylation level, also included in the Tissue lysates
Inhibitors of phosphatases and protease inhibitors, and the inhibitors of phosphatases and protease inhibitors be both needed to it is existing with existing plus；
The homogenization process carries out 30s on ice altogether, and Homogenization time is 10s every time, per interval between being homogenized twice
20s。
Preferably, in the assay method of described calpain phosphorylation level, in the step one, the phosphorylation
The cumulative volume of antibody and the protein sample containing the protease is 400-600 μ l, the albumen sample containing calpain
The overall reaction amount of albumen is 3500-5000 μ g in product.
Preferably, in the assay method of described calpain phosphorylation level, in the step one, the phosphorylation
The concentration of antibody is 0.2-0.3 μ g/ μ l, and the phospho-AB uses the general anti-Dan Ke of phosphorylation serine/threonine/tyrosine
Grand antibody.
Preferably, it is described to contain calcium in the step one in the assay method of described calpain phosphorylation level
The protein sample of protease is incubated 2-14h with phospho-AB in concussion at 4 DEG C, to form the immune complex.
Preferably, in the assay method of described calpain phosphorylation level, the animal muscle sample is derived from sheep.
The present invention at least includes following beneficial effect：
The method of the present invention can equally be detected by Western Immuno precipitation and immunoblot assay in postmortem muscle
Go out the calpain of phosphorylation, required muscle samples amount is few, and operating process is relatively simple, reproducible, detection gained band
Clearly, potential injury of the test method to human body is small, is μ-calpain phosphorylation levels in a kind of efficient measure postmortem muscle
Method.The Biochemical changes of phosphorylation calpain in ovine skeletal muscle can be researched and analysed, also may extend to using this method
The research of other animal muscle Physiology and biochemistries.
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
Fig. 1 is the image after the chloro- 1- naphthols development processes of 4- in the embodiment of the present invention 1；
Fig. 2 is the exposure image figure after ECL development processes in the embodiment of the present invention 2；
Fig. 3 is the pvdf membrane image after the ECL development processes in the embodiment of the present invention 2；
Fig. 4 is the exposure image figure after the ECL development processes in the embodiment of the present invention 3；
Fig. 5 is the exposure image figure after the ECL development processes in the embodiment of the present invention 4；
Fig. 6 is the exposure image figure after the ECL development processes in the embodiment of the present invention 5；
Fig. 7 is the Quantity One software analysis figures of exposure image figure in the embodiment of the present invention 5.
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 reagent and kit used in the present invention：
Tissue lysates：25mM bicines, 150mM sodium chloride, addition inhibitors of phosphatases (Roche, per 10ml
Addition 1) and protease inhibitors (Roche adds 1 per 50ml), pH 7.6；
Sarcoplasm extract：100mM Tris base, 10mM EDTA, 0.05% mercaptoethanol, pH8.3
Phospho-AB：Phospho-Ser/Thr/Tyr antibody, Abcam ab15556：
2 × reproducibility sample-loading buffer：10%SDS 4mL, glycerine 2mL, 0.5M Tris-HCl pH6.82mL, 1M DTT
2.0mL, bromophenol blue 0.01g：
8% separation gel:Distilled water 4.68ml, 1.5M Tris-Hcl pH8.82.5ml, 10%SDS 100 μ l, 30%
Acr-Bis(37.5:1) 2.67ml, TEMED 5 μ l, the μ l of 10% ammonium persulfate 50；
4% concentration glue：Distilled water 3.05ml, 0.5M Tris-HCl (pH6.8) 1.25ml, 10%SDS 50 μ l, 30%
Acr-Bis(37.5:1) 0.65ml, TEMED 5 μ l, the μ l of 10% ammonium persulfate 25；
TBS buffer solutions：Tris 1.211g, NaCl 8.76g, 1mol/L HCl adjust pH to 7.5；
Included in confining liquid：TBS 50ml/L, Tween2025 μ l/L, BSA 1.5g/L：
TBST1：Add 0.5mL Tween20 per 500mLTBS；
TBST2：Tris 3.0275g, NaCl 4.38g, Tween200.5mL, HCl tune pH are settled to 500mL to 7.5
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；
The kit used in Western Immuno precipitation test：Thermo#26146.
The present invention provides a kind of assay method of calpain phosphorylation level, including：
Step 1: the protein concentration of the protein sample containing calpain is adjusted into 5.83~12.5 μ g/ μ l, it is backward its
Middle addition phospho-AB, the overall reaction amount of the phospho-AB and the albumen in the protein sample containing calpain
Mass ratio be 1:1750~2500, then the protein sample containing calpain is incubated with phospho-AB in concussion at 4 DEG C
Educate, to form immune complex, then the calpain sample for obtaining phosphorylation is enriched with by Western Immuno intermediate processing；With
And,
Step 2: detecting the calcium albumen of the phosphorylation obtained in the step one by protein immunoblotting method
The phosphorylation level of enzyme sample.
In one of embodiment of the present invention, preferably, in the step one, passing through Western Immuno precipitation side
The specific steps for the calpain sample that method enrichment obtains the phosphorylation include：
1.1) Protein A/G agarose resins slurries are taken and make its absorption on centrifugal column, wherein, the Protein
The volume ratio of A/G agarose resins slurries and the calcium protein sample is 2~3:The institute added in 40~60, and each centrifugal column
The volume for stating Protein A/G agarose resin slurries is 20-30 μ l；
1.2) by the immune complex add by step 1.1) handle after be adsorbed with the Protein A/G agar
In the centrifugal column of glucoresin, and 1h is incubated in 4 DEG C of isothermal vibrations, centrifuges, then washed using IP lavation buffer solutions afterwards
The Protein A/G agarose resins three times, then wash with IP condition buffer solutions the Protein A/G agarose resins one
Secondary, what is retained on the last centrifugal column is the ternary of calpain, phospho-AB and Protein A/G agarose resins
Compound；With,
1.3) to step 1.2) in retention have reproducibility loading buffer added in the centrifugal column of the ternary complex
Liquid, and in being incubated 5min at 100 DEG C, 10000 × g centrifuges 1min at room temperature after cooling, and the liquid that flows through of gained is enrichment
The phosphorylation calpain sample.
In one of embodiment of the present invention, preferably, in the step 2, passing through protein immunoblotting
Method is detected during the phosphorylation level of the calpain sample of the phosphorylation, is resisted with the calpain reacts first
Body uses μ-calpain monoclonal antibodies, and the concentration of the first antibody is 1.4-2 μ g/ml.
In such scheme, preferably, in the step 2, the phosphorus is being detected by protein immunoblotting method
During the phosphorylation level of the calpain sample of acidifying, the secondary antibody combined with the first antibody uses anti-
Mouse IgG, the concentration of the secondary antibody is 0.1-0.3 μ g/ml.
In one of embodiment of the present invention, preferably, also including containing described in extraction before the step one
There is the method for the protein sample of calpain：
Animal muscle sample is gathered, and according to mass volume ratio 1:The animal muscle sample is added Tissue lysates by 6
Or in sarcoplasm extract, by the animal muscle sample and the Tissue lysates in being homogenized on ice to the animal muscle sample
Product and the Tissue lysates form homogeneous homogenate, then produce the homogenate in supernatant is collected by centrifugation at 4 DEG C
To the protein sample containing calpain；And all steps are in operation on ice or ice bath operation；
Wherein, the Tissue lysates are 25mM bicine and 150mM sodium chloride, the tissue comprising concentration
The pH of lysate is 7.6；
The sarcoplasm extract includes sulfydryl second of the concentration for 100mM Tris base, 10mM EDTA and 0.05%
Alcohol, the pH of the sarcoplasm extract is 8.3.
In such scheme, preferably, also including inhibitors of phosphatases and albumen enzyme level in the Tissue lysates
Agent, and the inhibitors of phosphatases and protease inhibitors be both needed to it is existing with existing plus；
The homogenization process carries out 30s on ice altogether, and Homogenization time is 10s every time, per interval between being homogenized twice
20s。
In one of embodiment of the present invention, preferably, in the step one, the phospho-AB and described
The cumulative volume of protein sample containing the protease is albumen in 400-600 μ l, the protein sample containing calpain
Overall reaction amount is 3500-5000 μ g.
In one of embodiment of the present invention, preferably, in the step one, the concentration of the phospho-AB
For 0.2-0.3 μ g/ μ l, the phospho-AB uses the general anti-monoclonal antibody of phosphorylation serine/threonine/tyrosine.
In one of embodiment of the present invention, preferably, in the step one, the egg containing calpain
White sample is incubated 2-14h with phospho-AB in concussion at 4 DEG C, to form the immune complex.
In one of embodiment of the present invention, preferably, the animal muscle sample is derived from sheep.
Following embodiments using Neofuge 15R types table-type high-speed refrigerated centrifuge, the unidirectional electrophoresis systems of Bio-Rad and
Bio-Rad wet method transferring film instrument.
Embodiment 1
A) the sheep longissimus dorsi muscle sample preparation Tissue Lysates of -80 DEG C of preservations in part are taken, BCA methods determine protein concentration；
B) SDS-PAGE electrophoresis：Using 8% separation gel and 4% concentration glue (Acr：Bis=37.5：1), gel is every
Hole adds 50 μ g protein samples；Electrophoresis initial parameter is set to 70V, sets voltage after protein band is completely into separation gel
It is set to 110V and proceeds electrophoresis, until Bromophenol Blue dye migrates and terminates electrophoresis to glue feather edge；
C) protein immunoblotting (IB)：Electrophoresis unloads offset plate after terminating, and gel is put into transferring film buffer solution and balanced；
Pvdf membrane is cut to after gel size activates 15s with pure methanol, and transferring film buffering is put into together with ready transferring film special filter paper
Balanced in liquid；With wet method transferring film technology, the ice bath 100min under 100V so that the protein band on gel is transferred to pvdf membrane
On；After transferring film terminates, pvdf membrane is washed with TBS buffer solutions three times, each 1min；
Pvdf membrane is put into the hybridization bag cut, adds 5ml confining liquids, at room temperature the concussion closing 1h on shaking table；
First antibody abbreviation primary antibody, primary antibody is incubated：μ-calpain the monoclonals of Abcam companies are added in 5ml confining liquids
Antibody (article No.：Ab49652), concentration is 1.4 μ g/ml；Hybridization bag is changed, by pvdf membrane and the 4 DEG C of mistakes of primary antibody solution prepared
Night is incubated；
Secondary antibody abbreviation secondary antibody, secondary antibody is incubated：Pvdf membrane is washed with TBST1 buffer solutions three times, each 10min；In 5ml
Add the anti-mouse IgG (article No. is A9044) of Sigma companies in confining liquid, concentration is 0.1 μ l/ml, molten as secondary antibody
Liquid is incubated 1h with the concussion of pvdf membrane room temperature；
Pvdf membrane is washed with TBST2 buffer solutions three times, each 10min utilizes the chloro- 1- naphthols development process colour developings of 4- afterwards.
Embodiment 2
A) the sheep longissimus dorsi muscle sample preparation Tissue Lysates of -80 DEG C of preservations in part are taken, BCA methods determine protein concentration；
B) SDS-PAGE electrophoresis：Using 8% separation gel and 4% concentration glue (Acr：Bis=37.5：1), gel is every
Hole adds 50 μ g protein samples；Electrophoresis initial parameter is set to 70V, sets voltage after protein band is completely into separation gel
It is set to 110V and proceeds electrophoresis, until Bromophenol Blue dye migrates and terminates electrophoresis to glue feather edge；
C) protein immunoblotting (IB)：Electrophoresis unloads offset plate after terminating, and gel is put into transferring film buffer solution and balanced；
Pvdf membrane is cut to after gel size activates 15s with pure methanol, and transferring film buffering is put into together with ready transferring film special filter paper
Balanced in liquid；With wet method transferring film technology, the ice bath 100min under 100V so that the protein band on gel is transferred to pvdf membrane
On；After transferring film terminates, pvdf membrane is washed with TBS buffer solutions three times, each 1min；
Pvdf membrane is put into the hybridization bag cut, adds 5ml confining liquids, at room temperature the concussion closing 1h on shaking table；
Primary antibody is incubated：μ-calpain monoclonal antibody (the article No.s of Abcam companies are added in 5ml confining liquids：
Ab49652), concentration is 1.4 μ g/ml；Hybridization bag is changed, by pvdf membrane and the 4 DEG C of night incubations of primary antibody solution prepared；
Secondary antibody is incubated：Pvdf membrane is washed with TBST1 buffer solutions three times, each 10min；Sigma is added in 5ml confining liquids
The anti-mouse IgG (article No. is A9044) of company, concentration is 0.1 μ l/ml, is shaken as two corresponding anti-solution and pvdf membrane room temperature
It is incubated 1h；
Pvdf membrane is washed with TBST2 buffer solutions three times, each 10min, the exposure colour developing of ECL development processes.
Embodiment 3
A) take out and weigh 0.5g under the muscle samples preserved at -80 DEG C, freezing state, centrifuge tube is filled to after the shape that is cut into small pieces
In；
B) add 6 times of volumes sarcoplasm extract (100mM Tris base, 10mM EDTA, 0.05% mercaptoethanol,
PH8.3), 30s (being often homogenized 10s interval 20s) is homogenized on ice；
C) homogenate 10,000 × g in 4 DEG C of centrifuges of low temperature centrifuges 35min；
D) supernatant is taken to dispense after skimming upper-layer fat layer；
E) quantification of protein：BCA methods, determine concentration and are saved backup after -80 DEG C；
F) SDS-PAGE electrophoresis：Using 8% separation gel and 4% concentration glue (Acr：Bis=37.5：1), gel is every
Hole adds 50 μ g protein samples；Electrophoresis initial parameter is set to 70V, sets voltage after protein band is completely into separation gel
It is set to 110V and proceeds electrophoresis, until Bromophenol Blue dye migrates and terminates electrophoresis to glue feather edge；
G) protein immunoblotting (IB)：Electrophoresis unloads offset plate after terminating, and gel is put into transferring film buffer solution and balanced；
Pvdf membrane is cut to after gel size activates 15s with pure methanol, and transferring film buffering is put into together with ready transferring film special filter paper
Balanced in liquid；With wet method transferring film technology, the ice bath 100min under 100V so that the protein band on gel is transferred to pvdf membrane
On；After transferring film terminates, pvdf membrane is washed with TBS buffer solutions three times, each 1min；
Pvdf membrane is put into the hybridization bag cut, adds 5ml confining liquids, at room temperature the concussion closing 1h on shaking table；
Primary antibody is incubated：μ-calpain monoclonal antibody (the article No.s of Abcam companies are added in 5ml confining liquids：
Ab49652), concentration is 1.4 μ g/ml；Hybridization bag is changed, by pvdf membrane and the 4 DEG C of night incubations of primary antibody solution prepared；
Secondary antibody is incubated：Pvdf membrane is washed with TBST1 buffer solutions three times, each 10min；Sigma is added in 5ml confining liquids
The anti-mouse IgG (article No. is A9044) of company, concentration is 0.1 μ l/ml, is shaken as two corresponding anti-solution and pvdf membrane room temperature
It is incubated 1h；
Pvdf membrane is washed with TBST2 buffer solutions three times, each 10min, the exposure colour developing of ECL development processes.
Embodiment 4
A) the sheep longissimus dorsi muscle sample preparation Tissue Lysates of -80 DEG C of preservations in part are taken, BCA methods determine protein concentration；
B) Western Immuno precipitation (IP)：Antigen-antibody shakes incubation 2h in 4 DEG C of constant-temperature metal baths, immune multiple to be formed
Compound；
Other immunoprecipitations, SDS-PAGE electrophoresis and protein immunoblotting experimental procedure and the method for the invention one
Cause, the exposure colour developing of ECL development processes.
Embodiment 5
A) the sheep longissimus dorsi muscle sample preparation Tissue Lysates of -80 DEG C of preservations in part are taken, BCA methods determine protein concentration；
B) Western Immuno precipitation (IP)：The concentration of sample protein is adjusted to 10 μ g/ μ l, the sample for taking 500 μ l deployed,
Add 2 μ g phospho-ABs (phospho-Ser/Thr/Tyr antibody, Abcam ab15556) so that antigen-antibody is anti-
The overall reaction amount for answering albumen in system is about 5000 μ g, and the concentration of phospho-AB is 0.2 μ g/ μ l；Antigen-antibody is in 4 DEG C of constant temperature
Concussion is incubated 14h in metal bath, to form immune complex；
The μ l of Protein A/G agarose resins slurries 20 are taken to add in centrifugal column to truncate pipette tips, 1000 × g, 4 DEG C of centrifugations
1min, discards percolation liquid；Resin is washed with the IP lavation buffer solutions of 100 μ l precoolings twice, every time with 1000 × g, 4 DEG C of bars
Discarded after part centrifugation and flow through liquid；500 μ l antigen antibody complex is added in the centrifugal column containing agarose resin, 4 DEG C
Concussion is incubated 1h in constant-temperature metal bath；1000 × g, 1min is centrifuged at 4 DEG C, reservation flows through liquid and (do not contain phosphorylation in theory
Albumen, is verified for follow-up detection)；Resin is washed with 200 μ l IP lavation buffer solutions three times, every time all 1000 after washing
× g, centrifuged under the conditions of 4 DEG C, discard and flow through liquid；100 μ l conditions buffer solutions wash resin once, 1000 × g, 4 DEG C of bars
Centrifuged under part, discard and flow through liquid, what is retained on centrifugal column is the ternary complex of antigen-antibody and agarose resin；
Added into ternary complex and be incubated 5min at 50 μ l SDS 2 × reproducibility sample-loading buffers, 100 DEG C, after cooling
10000 × g centrifuges 1min at room temperature, and gained flows through the phosphorylated protein sample that liquid is enrichment；
C) SDS-PAGE electrophoresis：Using 8% separation gel and 4% concentration glue (Acr：Bis=37.5：1), gel is every
Hole adds the phosphorylated protein sample of 20 μ l enrichments；Electrophoresis initial parameter is set to 70V, when protein band is completely into separation
Voltage is set to 110V after glue and proceeds electrophoresis, until Bromophenol Blue dye migrates and terminates electrophoresis to glue feather edge；
D) protein immunoblotting (IB)：Electrophoresis unloads offset plate after terminating, and gel is put into transferring film buffer solution and balanced；
Pvdf membrane is cut to after gel size activates 15s with pure methanol, and transferring film buffering is put into together with ready transferring film special filter paper
Balanced in liquid；With wet method transferring film technology, the ice bath 100min under 100V so that the protein band on gel is transferred to pvdf membrane
On；After transferring film terminates, pvdf membrane is washed with TBS buffer solutions three times, each 1min；
Pvdf membrane is put into the hybridization bag cut, adds 5ml confining liquids, at room temperature the concussion closing 1h on shaking table；
Primary antibody is incubated：μ-calpain monoclonal antibody (the article No.s of Abcam companies are added in 5ml confining liquids：
Ab49652), addition is 1.4 μ g/ml；Hybridization bag is changed, by pvdf membrane and the 4 DEG C of night incubations of primary antibody solution prepared；
Secondary antibody is incubated：Pvdf membrane is washed with TBST1 buffer solutions three times, each 10min；Sigma is added in 5ml confining liquids
The anti-mouse IgG (article No. is A9044) of company, concentration is 0.1 μ l/ml, is shaken as two corresponding anti-solution and pvdf membrane room temperature
It is incubated 1h；
Pvdf membrane is washed with TBST2 buffer solutions three times, each 10min, the exposure colour developing of ECL development processes.
As shown in figure 1, after protein immunoblotting experiment is developed the color with the chloro- 1- naphthols methods of 4-, except egg on pvdf membrane
Do not observe that any protein band occurs outside white Marker bands.And under the same terms, be exposed with ECL development processes aobvious
After color, as shown in Fig. 2 the protein band of three kinds of molecular weight of detectable very clear obvious calpain, and after exposure
Pvdf membrane correspondence fluorescent places have the appearance of dark yellow band, and Band signal is significantly stronger than Fig. 1.Illustrate in detection calpain band
During signal ECL development processes should be selected to be tested, in view of phosphorylation calpain Band signal is weaker, so present invention selection
ECL development processes as phosphorylation calpain imaging method.
Fig. 2 and Fig. 4 are respectively embodiment 2,3 experimental results, and Fig. 3 is shown after the ECL development processes in embodiment 2
Pvdf membrane image.In the case of other experiment condition identicals, embodiment 2 prepares laboratory sample using Tissue lysates, and
Embodiment 3 prepares sample using sarcoplasm extract.Two figures are contrasted it can be found that the band of calpain is deformed upon in Fig. 4, and
Do not possess the condition of subsequent data analysis processing.This is probably because the mercaptoethanol in extract disturbs normal SD S-PAGE
Being smoothed out for electrophoresis, causes protein band migration abnormal, therefore the present invention have selected tissue lysate method as the preparation side of sample
Method.
When the time that antigen-antibody shakes incubation is shorter, such as embodiment 4, shown in Fig. 5, after ECL exposure images, gained
Image background it is higher, the phosphorylation calpain bands of 1-4 swimming lane purposes is more obscured, analysis of experimental data can be produced compared with
Big interference.When antigen-antibody concussion incubation time extends to 14h, such as embodiment 5, shown in Fig. 6, image background is significantly lower than figure
5, and 3-4 swimming lane phosphorylation calpain bands are clearly obvious, beneficial to follow-up analyzing and processing.
The result of semi-quantitative analysis is carried out to the result of the test shown in Fig. 6 by Quantity One (Bio-Rad) softwares
As shown in fig. 7, the gray value of the calpain band of 3,4 swimming lane phosphorylations is respectively 781.018 and 339.986.Illustrate 3,4 swimming
In sample used in road there is notable difference, 3 Lane Sample μ-calpain phosphorylation levels in μ-calpain phosphorylation level
2.3 times of about 4 Lane Samples.And the tenderness of the meat of the level of calcium protein phosphorylation is related, by calcium protein phosphorylation
Level can also evaluate sample source Meat Tenderness.
Embodiment 6
A kind of assay method of calpain phosphorylation level, is carried out according to the following steps：
1) pig muscle sample is gathered, after liquid nitrogen flash freezer, is saved backup in less than -80 DEG C；
2) sample segment is taken to add Tissue lysates (25mM bicines, 150mM sodium chloride, the addition of 6 times of volumes
Inhibitors of phosphatases (Roche adds 1 per 10ml) and protease inhibitors (Roche adds 1 per 50ml), pH 7.6；
Inhibitors of phosphatases and protease inhibitors now need to be added with existing), 30s (being often homogenized 10s interval 20s) is homogenized on ice,；
3) homogenate centrifuges 15min under the conditions of 4 DEG C, 10,000g；
4) upper strata lipid is removed in drift after centrifuging, and Aspirate supernatant packing is stored for future use in -80 DEG C；Protein sample extracts the stage
Need whole operation on ice or suitably carry out ice bath.
5) quantification of protein：The protein concentration for extracting sample is determined using BCA methods (Thermo#23225)；
6) Western Immuno precipitation test (kit：Thermo#26146)：Phospho-AB (phospho-Ser/Thr/
Tyr antibody, Abcam ab15556) IP concentration be 0.3 μ g/ μ l, albumen is total in 600 μ l antigen-antibody reaction systems
The μ g of reacting dose about 3500, now protein concentration is 5.83 μ g/ μ l, and the concentration of phospho-AB is 3 μ g；Antigen-antibody is at 4 DEG C
Concussion is incubated 8h, to form immune complex；
The μ l of Protein A/G agarose resins slurries 30 are taken to add in centrifugal column to truncate pipette tips, 1000 × g centrifugations
1min, discards percolation liquid；Resin is washed with the IP lavation buffer solutions of 100 μ l precoolings twice, is discarded and is flowed through liquid；By antigen
Antibody complex is added in the centrifugal column containing agarose resin, and concussion is incubated 1h at 4 DEG C；1000 × g centrifuges 1min, retains stream
Wear liquid；Resin is washed with 200 μ l IP lavation buffer solutions three times, is all centrifuged after washing every time；100 μ l condition buffer solutions
Wash resin once；
Added in the ternary complex of gained and be incubated 5min at 50 μ l 2 × reproducibility sample-loading buffers, 100 DEG C,
10000 × g centrifuges 1min, and gained flows through the phosphorylated protein sample that liquid is enrichment.
7) SDS-PAGE electrophoresis：Using 8% separation gel and 4% concentration glue；
8) protein immunoblotting is tested：PAGE gel uses ice bath 100min under wet method transferring film, 100V, by gel
On protein band be transferred on pvdf membrane；TBS buffer solutions washing pvdf membrane three times, each 1min；
5ml confining liquids, room temperature concussion closing 1h are added in the hybridization bag equipped with pvdf membrane；Primary antibody is Abcam companies
μ-calpain monoclonal antibody (article No.s：Ab49652), concentration is 2 μ g/ml, 4 DEG C of night incubations；
TBST1 is washed three times, each 10min；Secondary antibody is the anti-mouse IgG (article No. is A9044) of Sigma companies,
Concentration is 0.3 μ l/ml, and room temperature concussion is incubated 1h；
TBST2 is washed three times, each 10min, the exposure colour developing of ECL (Bio-Rad#1705061) development process.
Wherein, the solution of whole process, which is prepared, need to use ultra-pure water.
Embodiment 7
A kind of assay method of calpain phosphorylation level, is carried out according to the following steps：
1) ox muscle samples are gathered, after liquid nitrogen flash freezer, are saved backup in less than -80 DEG C；
2) sample segment is taken to add Tissue lysates (25mM bicines, 150mM sodium chloride, the addition of 6 times of volumes
Inhibitors of phosphatases (Roche adds 1 per 10ml) and protease inhibitors (Roche adds 1 per 50ml), pH 7.6；
Inhibitors of phosphatases and protease inhibitors now need to be added with existing), 30s (being often homogenized 10s interval 20s) is homogenized on ice,；
3) homogenate centrifuges 15min under the conditions of 4 DEG C, 10,000g；
4) upper strata lipid is removed in drift after centrifuging, and Aspirate supernatant packing is stored for future use in -80 DEG C；Protein sample extracts the stage
Need whole operation on ice or suitably carry out ice bath.
5) quantification of protein：The protein concentration for extracting sample is determined using BCA methods (Thermo#23225)；
6) Western Immuno precipitation test (kit：Thermo#26146)：Phospho-AB (phospho-Ser/Thr/
Tyr antibody, Abcam ab15556) IP concentration be 0.3 μ g/ μ l, albumen is total in 400 μ l antigen-antibody reaction systems
The μ g of reacting dose about 5000, protein concentration is 12.5 μ g/ μ l, and the concentration of phospho-AB is 3 μ g；Antigen-antibody shakes at 4 DEG C
8h is incubated, to form immune complex；
The μ l of Protein A/G agarose resins slurries 20 are taken to add in centrifugal column to truncate pipette tips, 1000 × g centrifugations
1min, discards percolation liquid；Resin is washed with the IP lavation buffer solutions of 100 μ l precoolings twice, is discarded and is flowed through liquid；By antigen
Antibody complex is added in the centrifugal column containing agarose resin, and concussion is incubated 1h at 4 DEG C；1000 × g centrifuges 1min, retains stream
Wear liquid；Resin is washed with 200 μ l IP lavation buffer solutions three times, is all centrifuged after washing every time；100 μ l condition buffer solutions
Wash resin once；
Added in the ternary complex of gained and be incubated 5min at 50 μ l 2 × reproducibility sample-loading buffers, 100 DEG C,
10000 × g centrifuges 1min, and gained flows through the phosphorylated protein sample that liquid is enrichment.
7) SDS-PAGE electrophoresis：Using 8% separation gel and 4% concentration glue；
8) protein immunoblotting is tested：PAGE gel uses ice bath 100min under wet method transferring film, 100V, by gel
On protein band be transferred on pvdf membrane；TBS buffer solutions washing pvdf membrane three times, each 1min；
5ml confining liquids, room temperature concussion closing 1h are added in the hybridization bag equipped with pvdf membrane；Primary antibody is Abcam companies
μ-calpain monoclonal antibody (article No.s：Ab49652), concentration is 2 μ g/ml, 4 DEG C of night incubations；
TBST1 is washed three times, each 10min；Secondary antibody is the anti-mouse IgG (article No. is A9044) of Sigma companies,
Concentration is 03 μ l/ml, and room temperature concussion is incubated 1h；
TBST2 is washed three times, each 10min, the exposure colour developing of ECL (Bio-Rad#1705061) development process.
Wherein, the solution of whole process, which is prepared, need to use ultra-pure water.
Treatment scale described herein is the explanation for simplifying the present invention.Protein Extraction, phosphoric acid to the present invention
The application of change method and Western Immuno precipitation and the method such as Western blotting, modifications and variations are to one skilled in the art
It is obvious.
As described above, according to the present invention, detect that the calpain of phosphorylation in sheep longissimus dorsi muscle achieves success, and, warp
Test, the inventive method experimental repeatability is good, reliable results, can be examined for phosphorylation calpain in the animals skeletal muscles such as sheep repeatedly
Survey research and technical example is provided.
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 (8)
1. a kind of assay method of calpain phosphorylation level, including：
Step 1: the protein concentration of the protein sample containing calpain is adjusted into 5.83 ~ 12.5 μ g/ μ l, add thereto afterwards
Enter phospho-AB, the phospho-AB and the matter of the overall reaction amount of the albumen in the protein sample containing calpain
Amount is than being 1:1750 ~ 2500, then the protein sample containing calpain and phospho-AB are incubated in concussion at 4 DEG C, with
Immune complex is formed, then the calpain sample for obtaining phosphorylation is enriched with by Western Immuno intermediate processing；
Step 2: detecting the calpain sample of the phosphorylation obtained in the step one by protein immunoblotting method
The phosphorylation level of product；
The method for also including extracting the protein sample containing calpain before the step one：
Animal muscle sample is gathered, and according to mass volume ratio 1:6 add the animal muscle sample in Tissue lysates, will
The animal muscle sample and the Tissue lysates in being homogenized to the animal muscle sample and the Tissue lysates on ice
Homogeneous homogenate is formed, then obtains described containing calpain in supernatant is collected by centrifugation at 4 DEG C by the homogenate
Protein sample；And in addition to the step of animal muscle sample and the Tissue lysates are formed into homogenate, other own
Step is in operation on ice or ice bath operation；
Wherein, the Tissue lysates are 25 mM bicine and 150 mM sodium chloride comprising concentration, and the tissue splits
The pH for solving liquid is 7.6；
Inhibitors of phosphatases and protease inhibitors, and the inhibitors of phosphatases and albumen are also included in the Tissue lysates
Enzyme inhibitor is both needed to existing use and now added；
The homogenization process carries out 30 s on ice, and Homogenization time is 10 s every time, and 20 s are spaced per between homogenate twice.
2. the assay method of calpain phosphorylation level as claimed in claim 1, it is characterised in that in the step one,
The specific steps for being enriched with the calpain sample for obtaining the phosphorylation by Western Immuno intermediate processing include：
1.1）Take Protein A/G agarose resins slurries and make its absorption on centrifugal column, wherein, the Protein A/G
The volume ratio of agarose resin slurries and the protein sample containing calpain is 2 ~ 3:In 40 ~ 60, and each centrifugal column
The volume of the Protein A/G agarose resin slurries added is 20-30 μ l；
1.2）The immune complex is added and passes through step 1.1）The Protein A/G agarose trees are adsorbed with after processing
In the centrifugal column of fat, and 1h is incubated in 4 DEG C of isothermal vibrations, centrifuged afterwards, it is then described using the washing of IP lavation buffer solutions
Protein A/G agarose resins three times, then wash the Protein A/G agarose resins once with IP condition buffer solutions,
The ternary of the as calpain, phospho-AB and Protein A/G agarose resins that are retained on the last centrifugal column is answered
Compound；With,
1.3）To step 1.2）Middle retain adds reproducibility sample-loading buffer in the centrifugal column for having the ternary complex, and
In being incubated 5min at 100 DEG C, 10000 × g centrifuges 1min at room temperature after cooling, and the liquid that flows through of gained is the described of enrichment
The calpain sample of phosphorylation.
3. the assay method of calpain phosphorylation level as claimed in claim 1, it is characterised in that in the step 2,
It is and described during the phosphorylation level of calpain sample of the phosphorylation is detected by protein immunoblotting method
The first antibody of calpain reaction uses μ-calpain monoclonal antibodies, and the concentration of the first antibody is 1.4-2 μ g/
ml。
4. the assay method of calpain phosphorylation level as claimed in claim 3, it is characterised in that in the step 2,
It is and described during the phosphorylation level of calpain sample of the phosphorylation is detected by protein immunoblotting method
The secondary antibody that first antibody is combined uses anti-mouse IgG, and the concentration of the secondary antibody is 0.1-0.3 μ g/ml.
5. the assay method of calpain phosphorylation level as claimed in claim 1, it is characterised in that in the step one,
The cumulative volume of the phospho-AB and the protein sample containing calpain is 400-600 μ l, described to contain calcium albumen
The overall reaction amount of albumen is 3500-5000 μ g in the protein sample of enzyme.
6. the assay method of calpain phosphorylation level as claimed in claim 1, it is characterised in that in the step one,
The concentration of the phospho-AB is 0.2-0.3 μ g/ μ l, and the phospho-AB uses phosphorylation serine/threonine/junket
The general anti-monoclonal antibody of propylhomoserin.
7. the assay method of calpain phosphorylation level as claimed in claim 1, it is characterised in that in the step one,
The protein sample containing calpain is incubated the h of 2- 14 with phospho-AB in concussion at 4 DEG C, described immune multiple to be formed
Compound.
8. the assay method of calpain phosphorylation level as claimed in claim 1, it is characterised in that the animal muscle sample
Product are derived from sheep.
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