CN106085980A - Lactate dehydrogenase isoenzyme electrophoresis separating method - Google Patents
Lactate dehydrogenase isoenzyme electrophoresis separating method Download PDFInfo
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- CN106085980A CN106085980A CN201610414248.1A CN201610414248A CN106085980A CN 106085980 A CN106085980 A CN 106085980A CN 201610414248 A CN201610414248 A CN 201610414248A CN 106085980 A CN106085980 A CN 106085980A
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Abstract
The invention discloses a kind of lactate dehydrogenase isoenzyme separation method.The defect existed using barbital as the separation method of electrophoretic buffer for prior art, the invention provides a kind of lactate dehydrogenase isoenzyme electrophoresis separating method with tbe buffer liquid as electrophoretic buffer, and tbe buffer liquid is Tris 10.8g, Na2EDTA·2H2O 0.744g, boric acid 5.5g, add distilled water and dissolve, and is settled to 1000ml and prepares.The method can be optimized in terms of controlling electrophoresis temperature and/or controlling electrophoretic buffer pH two.The present invention also provides for the human tumor cell's lactate dehydrogenase isoenzyme electrophoresis separating method utilizing above-mentioned lactate dehydrogenase isoenzyme electrophoresis separating method to realize, and the application process that a kind of tbe buffer liquid is in lactate dehydrogenase isoenzyme is separated by electrophoresis.The inventive method effectively prevent the technological deficiency produced in prior art owing to barbitol buffer solution uses, and extends lactate dehydrogenase isoenzyme electrophoresis separating method using value in scientific research, Clinical detection, experimental teaching.
Description
Technical field
The present invention relates to a kind of enzyme electrophoresis separating method, particularly relate to a kind of lactate dehydrogenase isoenzyme electrophoretic separation side
Method.Belong to tissue and cytoactive enzyme electrophoresis detection field
Background technology
Lactate dehydrogenase isoenzyme (lactate dehydrogenase, LDH) is to generate third by reversible catalysis lactic dehydrogenase
The enzyme of keto acid, metabolism and energy supply with sugar have important relationship.
Lactic acid dehydrogenase is widely present in each histiocytic endochylema of health, the abundantest with the heart, skeletal muscle and kidney, its
Secondary for liver, spleen, pancreas, brain and lung etc..It is similar that it comprises 5 be made up of respectively A subunit (skeletal muscle type) and B subunit (cardiac muscle type)
Work enzyme, i.e. LDH1 (B4), LDH2 (B3A), LDH3 (B2A2), LDH4 (BA3), LDH5 (A4).LDH isozyme is in different tissues
, there is obvious tissue specificity in distribution difference.Based on LDH1, LDH2 in the heart, kidney, erythrocyte, in liver, skeletal muscle with
LDH4, LDH5 are main, and lung, spleen, pancreas, thyroid, adrenal gland and lymph node etc. are most with LDH3 in organizing.Owing to LDH is significant
Tissue specificity, therefore LDH isozyme related experiment becomes medical speciality Theory Course and laboratory selective learning content.
Being present in various cell of LDH isozyme quite stable under normal circumstances, but when different tissues destroys impaired,
Different isozymes is released into blood, and after showing effect such as acute myocardial infarction, in early stage blood serum, LDH1 and LDH2 activity all raises, but
LDH1 increases earlier, becomes apparent from, and causes the ratio of LDH1/LDH2 to raise；When hepatitis, acute hepatocellular injury and Skeletal muscle injury
LDH5 can raise；Suffer from the sick LDH1 such as activeness rheumatic heart disease, acute viral myocarditis, hemolytic anemia, renal necrosis
Also can raise etc..Therefore diagnosis can be made according to the change of various isozymes activity.So, lactate dehydrogenase isoenzyme
Detection by quantitative is the most significant in terms of Clinical Laboratory.
In terms of basic research, it is the most relevant to tumor evolution, as at mammary gland that existing result of study has proven to LDH isozymogram
Cancerous cell suppresses LDHB rush tumor effect then occurs, suppress LDHA that obvious Graft Versus Tumor then occurs.Meanwhile, body
Interior experiment display, LDHA inhibitor is at antineoplastic simultaneously almost without cytotoxicity, and in terms of clinical research, some are popular
The sick investigation learned, it was also found that obvious disease can't occur in the ethnic group lacking LDHA, only produces flesh after violent anaerobic exercise
Red eggs albiduria.Therefore, LDH and isozyme thereof are expected to become the specificity antineoplastic molecular target that side effect is little.
To sum up, the detection by quantitative of lactate dehydrogenase isoenzyme all has in terms of experimental teaching, Clinical Laboratory, scientific research
Suitable using value and wide application prospect.
Prior art generally uses electrophoretic separation technique (cellulose acetate membrane electrophoresis, agarose gel electrophoresis) to measure and divides
From 5 kinds of LDH isozymes, and using barbital as electrophoretic buffer.At least there is the deficiency of four aspects in the method: one, in recent years
Barbital and barbital sodium are increasingly becoming national regulatory medicine, buy more and more difficult, and price is costly；Two, barbital buffering
No matter liquid is all difficult to preserve at room temperature or low temperature；When three, using barbitol buffer solution to be separated by electrophoresis, heating is serious, easily leads
Cause experimental result band the best；Four, prior art range of application in terms of separating and detecting lactate dehydrogenase isoenzyme is minimum:
On the one hand due to the particularity of different plant species isoelectric point, IP, prior art can the agarose gel method of quantitative analysis be only applicable to little
This kind of isoelectric point, IP of Mus, at narrower and moderate species, is unsuitable for the species of human tumor cell's this kind of isoelectric point, IP wider range.Separately
Though on the one hand acetate film method is not limited by isoelectric point, IP, but sensitivity is low, can not be quantitative, is only applicable to routine experimentation religion
Learn.
Summary of the invention
The purpose of the present invention is aiming at the deficiencies in the prior art, it is provided that a kind of new lactate dehydrogenase isoenzyme electrophoresis divides
From method, the method uses new electrophoretic buffer, it is possible to be applicable to agarose gel electrophoresis separation method thin with acetate fiber
Membrane electrophoresis separation method, and each species lactic acid dehydrogenase being used for including human tumor cell's lactate dehydrogenase isoenzyme
The electrophoretic separation of isozyme and quantitative analysis.
For achieving the above object, present invention firstly provides a kind of lactate dehydrogenase isoenzyme electrophoresis separating method, its technology
Scheme is as follows:
A kind of lactate dehydrogenase isoenzyme electrophoresis separating method, it is characterised in that: electrophoretic buffer is tbe buffer liquid, institute
Stating tbe buffer liquid is Tris 10.8g, Na2EDTA·2H2O 0.744g, boric acid 5.5g, add distilled water and dissolve, be settled to
1000ml prepares.
Through the verification experimental verification of different plant species histiocyte material, tbe buffer liquid can be as electrophoretic buffer for multiple
The electrophoretic separation of the lactate dehydrogenase isoenzyme in animal, human tissue cell, divides LDH1, LDH2, LDH3, LDH4, LDH5
From effective.
Said method can be not only used for agarose gel electrophoresis separation method it can also be used to cellulose acetate membrane electrophoresis separation side
Method.
When said method is applied to agarose gel electrophoresis separation method, two aspect conditions can be used further to control to add
To optimize: one is to control electrophoresis temperature constant temperature 10 DEG C～12 DEG C；Two according to detected species lactate dehydrogenase isoenzyme
Isoelectric point, IP, controls electrophoretic buffer pH at optimum range.Two kinds of optimal conditions control all there is independent effect.
Based on above-mentioned lactate dehydrogenase isoenzyme electrophoresis separating method, present invention simultaneously provides a kind of human tumor cell's breast
Acidohydrogenase isozyme electrophoresis separation method, it is possible to achieve lactate dehydrogenase isoenzyme in human tumor cell is efficiently separated
Measuring, its technical scheme is as follows:
A kind of human tumor cell's lactic acid dehydrogenase utilizing above-mentioned lactate dehydrogenase isoenzyme electrophoresis separating method to realize
Isozyme electrophoresis separation method.
The present invention also provides for a kind of tbe buffer liquid application process in lactate dehydrogenase isoenzyme is separated by electrophoresis, its skill
Art scheme is as follows:
The application in lactate dehydrogenase isoenzyme is separated by electrophoresis of the tbe buffer liquid, described tbe buffer liquid is Tris
10.8g、Na2EDTA·2H2O 0.744g, boric acid 5.5g, add distilled water and dissolve, and is settled to 1000ml and prepares.
Compared with prior art, the invention has the beneficial effects as follows: (1) the invention provides a kind of using tbe buffer liquid as
The electrophoresis method of electrophoretic buffer separating lactic acid dehydrogenase isoenzyme, is a kind of brand-new separation detection lactate dehydrogenase isoenzyme
Electrophoresis method；(2) in the agarose gel electrophoresis separation method using tbe buffer liquid as electrophoretic buffer, the present invention is led to
Basic technical scheme is optimized by the means such as control electrophoresis temperature or control electrophoretic buffer pH value of crossing, and improves separation effect
Really；(3) the inventive method can be applied to the electrophoresis separating method of human tumor cell's lactate dehydrogenase isoenzyme, extends breast
Acidohydrogenase isozyme electrophoresis separation method using value in scientific research and Clinical detection；(4) the invention provides one
Plant human tumor cell's lactate dehydrogenase isoenzyme electrophoresis separating method, it is possible to stablized and good experimental result, and
Quantitative analysis can be carried out, overcome existing electrophoresis method experimental result when separating human tumor cell's lactate dehydrogenase isoenzyme
Unstable, experimental result is poor, be difficult to the defect of data analysis；(5) the invention provides tbe buffer liquid at lactic dehydrogenase
Application process in enzyme isoenzyme electrophoretic separation；(6) the tbe buffer liquid reagent that the present invention uses is conventional, be easy to get, and prepares same volume
Buffer cost is only same volume barbitol buffer solution 1/10, simultaneously after configuration room temperature be unlikely to deteriorate when preserving, can be repeated multiple times
Reclaiming and use, in electrophoretic procedures, buffer heating is slightly slight, and a whole set of electrophoretic procedures step is simple, condition is easily-controllable, result is stable,
Can solve the problem that the heating occurred in barbitol buffer solution electrophoresis is high, effect unstable, room temperature or cryopreservation is the most perishable etc. lacks
Fall into.
Accompanying drawing explanation
Fig. 1 .1 is that agarose gel electrophoresis method separates different human tumor cell LDH isozyme electrophoresis collection of illustrative plates (1.BT-
549,2.ZR-75-30,3.A375,4.MDA-MB-231).
Fig. 1 .2 is the data analysis figure that agarose gel electrophoresis method separates different human tumor cell's LDH isozyme
(1.BT-549,2.ZR-75-30,3.A375,4.MDA-MB-231).
Fig. 2 .1 is that agarose gel electrophoresis method separates rat different tissues LDH isozyme electrophoresis collection of illustrative plates (the 1. heart, 2. liver, 3.
Kidney, 4. lung, 5. intestinal, 6. skeletal muscle).
Fig. 2 .2 is data analysis figure (the 1. heart, 2. that agarose gel electrophoresis method separates rat different tissues LDH isozyme
Liver, 3. kidney, 4. lung, 5. intestinal, 6. skeletal muscle).
Fig. 3 .1 is agarose gel electrophoresis method separating guinea pig different tissues LDH isozyme electrophoresis collection of illustrative plates (the 1. heart, 2. liver, 3.
Kidney, 4. lung, 5. intestinal, 6. skeletal muscle).
Fig. 3 .2 is data analysis figure (the 1. heart, 2. of agarose gel electrophoresis method separating guinea pig different tissues LDH isozyme
Liver, 3. kidney, 4. lung, 5. intestinal, 6. skeletal muscle).
Fig. 4 .1 is with tbe buffer liquid row cellulose acetate membrane electrophoresis separation different tissues or cell LDH isozyme electrophoresis figure
Spectrum (1. mankind mastopathy cell MDA-MB-231,2. the rat heart, 3. Guinea pig lung).
Temperature control and non-temperature control contrast knot when Fig. 5 .1 is agarose gel electrophoresis separation MDA-MB-231 cell LDH isozyme
Really (1. temperature control, 2. without temperature control).
The comparing result of difference pH when Fig. 6 .1 is agarose gel electrophoresis separation MDA-MB-231 cell LDH isozyme
(1.pH8.3、2.pH8.0)
Detailed description of the invention
Below in conjunction with the accompanying drawings, the preferred embodiments of the present invention are further described.
Embodiment one
In different human tumor cells, lactate dehydrogenase isoenzyme agarose gel electrophoresis separates.
1, material
1.1 equipment
Centrifuge, constant temperature cell culture incubator, inverted microscope, superclean bench, Tissue Culture Flask, cytology brush, pipet,
Agarose gel Horizontal electrophoresis tank, electrophresis apparatus, glue mould, constant water bath box, baking box, electronic balance, agitator, microwave oven,
Gel imaging system, EP pipe, liquid-transfering gun, pH meter
1.2 preparation of reagents
Phosphate buffer (PBS): Na2HPO4.7H2O 22.55g、NaH2PO42.16g, adds distilled water and dissolves, be settled to
1000ml, pH 7.4；
Tryptic digestive juice: 0.5g Trypsin, 0.04g Na2EDTA·2H2O, is dissolved in PBS, adds PBS and is settled to
200ml, pH 7.2；
10 times concentrate TBE (Tris-Boric acid-EDTA) buffer: Tris 108g, Na2EDTA·2H2O 7.44g、
Boric acid 55g, adds distilled water and dissolves, be settled to 1000ml；1 times of concentration it is diluted to (such as: preparation 1 times of TBE of 1000ml delays during use
Rush liquid, i.e. take 10 times of tbe buffer liquid of 100ml and add distilled water and be settled to 1000ml), regulate pH to 8.3；Hereinafter referred to as tbe buffer
Liquid；
1mol/L sodium lactate: 60% sodium lactate solution 9.25ml, adds distilled water and is settled to 50ml, 4 DEG C of preservations；
Normal saline: NaCl 0.584g, adds distilled water and dissolves, be settled to 100ml；
1mg/ml phenazine methosulfate (PMS): PMS 100mg, adds distilled water and dissolves, be settled to 100ml, brown bottle 4
DEG C preserve；
1mg/ml NBT (NBT): NBT 100mg, adds distilled water and dissolves, be settled to 100ml, brown bottle
4 DEG C of preservations；
NAD+ (nicotinamide adenine dinucleotide): analytical pure
1 ‰ Triton X-100:Triton X-100 1ml, add distilled water, are settled to 1000ml；
10 times of concentration sample-loading buffers: bromophenol blue 0.04g, 1 times of tbe buffer liquid 5ml, sucrose 10g, heat, dissolve, cooling
To room temperature, add 1 times of tbe buffer liquid be settled to 10ml.
DMEM culture medium: one bag of DMEM powder (is bought from gibco by life technologies), adds ultra-pure water
700ml (15～30 DEG C), stirring and dissolving, add 3.7gNaHCO3, stirring and dissolving, regulate pH to 7.2, add ultra-pure water and be settled to
900ml, filtration, subpackage to 250ml reagent bottle, culture plate spot hole is put into incubator, is checked whether to pollute, add 10% tire during use
Ox blood serum (as preparation 100mlDMEM culture medium i.e. 90mlDMEM solution adds 10ml hyclone)
1640 culture medium: modified form RPMI-1640 culture medium (is bought and flown your biochemistry goods of generation (Beijing) in Sai Mo and have
Limit company) add 10% hyclone (as preparation 100ml1640 culture medium i.e. 90ml modified form RPMI-1640 culture medium adds
10ml hyclone)
Hyclone: buy and fly your biochemistry goods (Beijing) company limited of generation in Sai Mo
Dyeing liquor: include the economic dyeing liquor of low concentration rapid dye liquor, low concentration, optional one during use.Formula is as follows:
Table 1.1 low concentration rapid dyeing formula of liquid
The economic prescription of its dyeing liquor of table 1.2 low concentration
1.3 samples and preparation
1.3.1 cell material
Different human tumor cells: BT-549, ZR-75-30, A375, MDA-MB-231
1.3.2 cell cultivates (seeing reference paper 1)
By appropriate cell material holding in 50 milliliters of specification culture bottles, 37 DEG C, 5%CO2, cultivate under saturated humidity environment,
Change liquid after 2d～3d, put into and continue under same environment to cultivate；Until cell length at the bottom of accounting for bottle about 80% time Secondary Culture.Pass on behaviour
Concretely comprise the following steps: suck original fluid, wash 1 time with PBS, thin with the digestion of 1ml pipette, extract 1 pipe tryptic digestive juice
Born of the same parents；In basis of microscopic observation, when cell starts substantially to shrink, discard pancreatin, then with pipette, extract 2 pipe culture medium (ZR-
75-30, A375, MDA-MB-231 cell line uses DMEM culture medium, and BT-549 uses 1640 culture medium), blow and beat into unicellular
Suspension, subpackage to two 50ml specification culture bottle, supplements the 2 corresponding DMEM of pipet or 1640 culture medium respectively, is placed in above-mentioned
37 DEG C, 5%CO2, cultivate under saturated humidity environment.General Secondary Culture to the 3rd generation can be used in prepares cell sample.
1.3.3 prepared by cell sample
When cell length to 90% at the bottom of culture bottle bottle～95%, take each one bottle of four kinds of cells (50ml specification), carry out respectively
Following operation: discard culture supernatant, PBS washs 3 times；Add 0.5ml 1 ‰ Triton X-100, scraping cells suspension to EP
Pipe, 37 DEG C of constant temperature incubators hatch 30min；8 DEG C, 16000r/min be centrifuged 30min, take supernatant, add 10 times concentrate loading buffer
Liquid is (such as: take supernatant 180 μ l and then add 10 times of concentrations sample solution 20 μ l, altogether 200 μ l, i.e. concentrate sample solutions with supernatant by 10 times
It is diluted to 1 times), softly blow and beat mixing with rifle head, room temperature preservation is stand-by.
2, agarose gel electrophoresis lock out operation
Prepared by 2.1 agarose gel (5g/L)
This experiment mould is 6cm × 12cm that 1.5mm is thick, and 6 hole combs, gel strength is 0.5%.
Cleaning glue mould and comb, be placed on balancer, and inserted in by comb on mould after drying, comb is away from mould
Minor face about 2.5cm.Weigh 0.15g agarose gel particle, pour in conical flask, separately measure tbe buffer liquid 30ml and add, Yu Wei
In ripple stove, glue 1min is boiled in moderate heat heating, takes out, the most softly shakes up, observing with or without undissolved granule, if having, then repeating to boil glue
Once.After gel is completely dissolved, adds tbe buffer liquid, is settled to 30ml.30ml gel solution is poured slowly into glue grinding tool
In, gel 60min.
2.2 electrophoresis
Clean electrophoresis tank, pour the tbe buffer liquid of pH 8.3, the comb that takes out the slowest, parallel into, take out gel slab, put
Enter electrophoresis tank, slowly add electrophoresis liquid to submergence gel upper surface.Take the four kinds of cell samples prepared in step 1.3.3 respectively
It is appropriate (determining applied sample amount according to conventional practices, loading 5 μ l～30 μ l in the present embodiment) from, loading in desk centrifuge point,
Keeping electrophoresis liquid temperature constant at 10 DEG C～12 DEG C, constant voltage 100V, to bromophenol blue index strip arrival glue afterbody (about 180min altogether).
2.3 dyeing and imagings
Electrophoresis takes out gel after completing and dyes in 37 DEG C of constant water bath box 5min～10min (the quick formula of low concentration),
Or 40min～60min (low concentration economical formula) (concrete dyeing time is clear with the colour developing of each band, and clear background is advisable) and becomes
Picture, analysis.
3, separating resulting
Fig. 1 .1 is that agarose gel electrophoresis method separates different human tumor cell LDH isozyme electrophoresis collection of illustrative plates (1.BT-
549,2.ZR-75-30,3.A375,4.MDA-MB-231), Fig. 1 .2 is that the different human tumor of agarose gel electrophoresis method separation is thin
The data analysis figure (1.BT-549,2.ZR-75-30,3.A375,4.MDA-MB-231) of born of the same parents' LDH isozyme.Data analysis process
Carry out gray analysis with ImageJ, carry out icon drafting with CAD software and show various LDH isozymes according to graphics area conversion
Content.Fig. 1 .1 display uses the agarose gel electrophoresis method optimized to separate different human tumor cell's LDH isozyme and all separates effect
Fruit is good, and band is good, and Fig. 1 .2 display uses the agarose isozyme spectrogram of the technical program gained, can carry out the most quantitatively
Analyze.In different human tumor cells, the distribution of each lactate dehydrogenase isoenzyme is different.
Reference paper 1: Si Tuzhenqiang, Wu Junzheng. cell is cultivated. and world book publishes Xi'an company .2007.01
Embodiment two
In rat different tissues, lactate dehydrogenase isoenzyme agarose gel electrophoresis separates.
It does not repeats with embodiment one something in common, and its difference is:
1, material
1.1 equipment
Separately need homogenizer；It is not required to constant temperature cell culture incubator, inverted microscope, superclean bench, Tissue Culture Flask, cell
Brush, pipet, constant water bath box, baking box
1.2 preparation of reagents
10 times concentrate TBE (Tris-Boric acid-EDTA) buffer: formulation operations is with embodiment one, and regulation pH is extremely
8.0；
It is not required to tryptic digestive juice, 1 ‰ Triton X-100, DMEM culture medium, 1640 culture medium (remaining same embodiment
One).
1.3 samples and preparation
1.3.1 laboratory animal
Rat
1.3.2 acquisition (need not cell cultivate) is organized
3% anesthetics is prepared (such as: 10ml normal saline adds amobarbital with normal saline and amobarbital
0.3g), it is injected in laboratory animal intraperitoneal by 1ml/kg, after Animal Anesthesia, is allowed to dry systemic blood, reselection with carotid artery
Obtain tissue (such as: the heart, liver, kidney, lung, small intestinal, skeletal muscle etc.) and be immersed in normal saline standby.
1.3.3 tissue sample prepares (need not cell sample prepare)
Take 1.5ml EP pipe one, add 0.5ml normal saline with liquid-transfering gun, add Semen Glycines size single organization block, use
Small size tissue shear shreds, then is allowed to thoroughly grind by homogenizer；8 DEG C, 16000r/min be centrifuged 30min, take supernatant, add 10 ×
Concentrating sample-loading buffer, softly blow and beat mixing with rifle head, room temperature preserves stand-by.
2, agarose gel electrophoresis lock out operation
Prepared by 2.1 agarose gel (5g/L)
This experiment mould is 12 × 12cm that 1.5mm is thick, and 13 hole combs, gel strength is 0.5%.
Remaining with embodiment one.
2.2 electrophoresis
Clean electrophoresis tank, pour the tbe buffer liquid of pH8.0, the comb that takes out the slowest, parallel into, take out gel slab, put into
Electrophoresis tank, slowly adds electrophoresis liquid to submergence gel upper surface.Take the rat tissue's sample prepared (i.e. 1.3.3 to prepare
Rat tissue's sample) appropriate (with embodiment one) from, loading in desk centrifuge point, keep electrophoresis liquid temperature constant at 10 DEG C
～12 DEG C, constant voltage 100V, arrive glue afterbody (about 180min altogether) to bromophenol blue index strip.
3, separating resulting
Fig. 2 .1 is that agarose gel electrophoresis method separates rat different tissues LDH isozyme electrophoresis collection of illustrative plates (the 1. heart, 2. liver, 3.
Kidney, 4. lung, 5. intestinal, 6. skeletal muscle), Fig. 2 .2 is the data that agarose gel electrophoresis method separates rat different tissues LDH isozyme
Analysis chart (the 1. heart, 2. liver, 3. kidney, 4. lung, 5. intestinal, 6. skeletal muscle).Data analysis process carries out gray analysis with ImageJ, with
CAD software carries out icon drafting.It is same that Fig. 2 .1 display uses the agarose gel electrophoresis method optimized to separate rat different tissues LDH
The equal good separation of work enzyme, band is good, and Fig. 2 .2 display uses the agarose isozyme spectrogram of the technical program gained, can enter
The accurate quantitative analysis of row.In rat different tissues, the distribution of each lactate dehydrogenase isoenzyme is different.
Embodiment three
In Cavia porcellus different tissues, lactate dehydrogenase isoenzyme agarose gel electrophoresis separates.
It does not repeats with embodiment two something in common, and its difference is:
1, material
1.1 equipment (with embodiment two)
1.2 preparation of reagents
10 times concentrate TBE (Tris-Boric acid-EDTA) buffer: formulation operations is with embodiment one, and regulation pH is extremely
9.2。
1.3 samples and preparation
1.3.1 laboratory animal
Cavia porcellus 1.3.2 tissue obtains (with embodiment two, it is not necessary to cell is cultivated)
1.3.3 tissue sample prepares (with embodiment two, it is not necessary to prepared by cell sample)
2, agarose gel electrophoresis lock out operation
2.1 agarose gel (5g/L) prepare (with embodiment two)
2.2 electrophoresis
Clean electrophoresis tank, pour the tbe buffer liquid of pH9.2, the comb that takes out the slowest, parallel into, take out gel slab, put into
Electrophoresis tank, slowly adds electrophoresis liquid to submergence gel upper surface.Take the guinea pig tissues sample for preparing in desk centrifuge point from,
Loading appropriate (with embodiment one), keep electrophoresis liquid temperature constant at 10 DEG C～12 DEG C, constant voltage 100V, take to bromophenol blue instruction
Reach glue afterbody (about 210min altogether).
3, separating resulting
Fig. 3 .1 is agarose gel electrophoresis method separating guinea pig different tissues LDH isozyme electrophoresis collection of illustrative plates (the 1. heart, 2. liver, 3.
Kidney, 4. lung, 5. intestinal, 6. skeletal muscle), Fig. 3 .2 is the data of agarose gel electrophoresis method separating guinea pig different tissues LDH isozyme
Analysis chart (the 1. heart, 2. liver, 3. kidney, 4. lung, 5. intestinal, 6. skeletal muscle).Data analysis process carries out gray analysis with ImageJ, with
CAD software carries out icon drafting.Fig. 3 .1 display uses the agarose gel electrophoresis method separating guinea pig different tissues LDH optimized same
The equal good separation of work enzyme, band is good, and Fig. 3 .2 display uses the agarose isozyme spectrogram of the technical program gained, can enter
The accurate quantitative analysis of row.In Cavia porcellus different tissues, the distribution of each lactate dehydrogenase isoenzyme is different.
Embodiment four
In different tissues or cell, lactate dehydrogenase isoenzyme carries out cellulose acetate membrane electrophoresis separation with TBE electrophoresis liquid.
It does not repeats with embodiment one, two something in common, and its difference is:
1, material
1.1 equipment
Separately need homogenizer, cytology brush, preservative film, vinegar fibre film, filter paper；
Remaining with embodiment one.
1.2 preparation of reagents
10 times concentrate TBE (Tris-Boric acid-EDTA) buffer: formulation operations is with embodiment one, it is not necessary to regulation
pH。
Remaining with embodiment one.
1.3 samples and preparation
1.3.1 cell material and laboratory animal
Mankind mastopathy cell MDA-MB-231, rat, Cavia porcellus
1.3.2 acquisition (with embodiment two) is organized
1.3.3 tissue sample prepares (with embodiment two)
2, cellulose acetate membrane electrophoresis lock out operation
Prepared by 2.1 electrophoresis acetate films
Prepare 6cm × 3cm acetate film bar or the full film of acetate film of 6cm × 8cm, at 1 times of tbe buffer liquid
In (without regulating pH) invade bubble overnight.
2.2 electrophoresis
Clean electrophoresis tank, pour 1 times of tbe buffer liquid (without regulating pH) into, take salt bridge with four metafiltration paper.Take the acetic acid of immersion
Fiber membrane one, draws film surface excessive moisture with filter paper, is still on filter paper standby.20 μ l samples are drawn with liquid-transfering gun
Product, evenly laid out on preservative film, then dip sample with cytology brush, select in vinegar fibre film frosted face, away from film leading edge 1.5cm
Place, treats that sample all penetrates in film, removes cytology brush.By the thin film of point sample as on filter paper bridge, point sample faces down, point sample end
As for negative electrode.Constant voltage 150V, electrophoresis 30min, it is not necessary to control temperature, room temperature places electrophoresis.
2.3 dyeing and imagings
Electrophoresis complete after take out acetate film, point sample towards upper, in 37 DEG C of constant water bath box dye 5min～
10min (the quick formula of low concentration), or 40min～60min (low concentration economical formula) (the reagent dyeing time is clear with the colour developing of each band
Chu, clear background is advisable) and imaging, analysis.
3, separating resulting
Fig. 4 .1 is with tbe buffer liquid row cellulose acetate membrane electrophoresis separation different tissues or cell LDH isozyme electrophoresis figure
Spectrum (1. mankind mastopathy cell MDA-MB-231,2. the rat heart, 3. Guinea pig lung).Fig. 4 .1 display MDA-MB-231, the rat heart,
The equal good separation of Guinea pig lung, band is good, thus illustrates that carrying out cellulose acetate membrane electrophoresis with tbe buffer liquid separates the most of the same race
The LDH isozyme belonging to sample still can reach good result.
Embodiment five
Electrophoresis temperature controls controlled trial.It does not repeats with embodiment one something in common, and its difference is:
1, material
1.1 equipment (with embodiment one)
1.2 preparation of reagents (with embodiment one)
1.3 samples and preparation (with embodiment one)
1.3.1 cell material (with embodiment one)
1.3.2 cell cultivates (with embodiment one)
1.3.3 cell sample prepares (with embodiment one)
Prepared by 2.1 agarose gel (5g/L)
This experiment mould is 6cm × 12cm that 1.5mm is thick, and 6 hole combs, gel strength is 0.5%.
Clean two set glue mould and 6cm × 12cm comb, be placed on balancer after drying, and comb is inserted in mould
On, comb is away from mould minor face about 2.5cm.Weigh 0.3g agarose gel particle, pour in conical flask, separately measure tbe buffer liquid
60ml adds, and in microwave oven, glue 1min is boiled in moderate heat heating, takes out, the most softly shakes up, observes with or without undissolved granule, if
Have, then repeat to boil glue once.After gel is completely dissolved, adds tbe buffer liquid, is settled to 60ml.30ml gel is measured with graduated cylinder
Solution is poured slowly in the most a set of glue mould, and 30ml gel solution remaining in conical flask is poured slowly into another set of glue
In mould, gel 60min.
2.2 electrophoresis
Clean two set electrophoresis tanks, label " temperature control group ", " non-temperature control group ", pour the tbe buffer liquid of identical pH8.3 respectively into.
The comb taking out two set glue moulds the slowest, parallel, takes out gel slab, is arbitrarily each placed in one of them electrophoresis tank, slow
Slowly electrophoresis liquid is added to submergence gel upper surface.Take four kinds of cell sample (the i.e. cells prepared in 1.3.3 prepared respectively
Sample) appropriate (with embodiment one) from, loading in desk centrifuge point." temperature control group " keeps electrophoresis liquid constant temperature 10 DEG C～12 DEG C,
" non-temperature control group " is refused temperature and is interfered, and is still in indoor, two groups of equal constant voltages 100V, arrives glue afterbody (altogether to bromophenol blue index strip
About 120～180min, " temperature control group " about needs 180min, and " non-temperature control group " about needs 120min, and the concrete time is with bromophenol blue index strip
Arrive glue afterbody to be as the criterion).
3, separating resulting
Temperature control and non-temperature control contrast knot when Fig. 5 .1 is agarose gel electrophoresis separation MDA-MB-231 cell LDH isozyme
Really (1. temperature control, 2. without temperature control).By temperature control group LDH2 seen from Fig. 5 .1～LDH5 (MDA-MB-231 cell LDH1 expresses denier,
Generally can't detect) all separating effects are good, and band is good；Non-temperature control group LDH5 inactivates, is only capable of LDH2～LDH4 being detected.
Embodiment six
TBE (Tris-Boric acid-EDTA) pH of cushioning fluid controls controlled trial.It is with embodiment one something in common
Not repeating, its difference is:
1, material
1.1 equipment (with embodiment one)
1.2 preparation of reagents
10 times concentrate TBE (Tris-Boric acid-EDTA) buffer: formulation operations is with embodiment one, and regulation pH is extremely
8.3；1 times of tbe buffer liquid that another preparation is identical, regulates pH to 8.0.
Remaining with embodiment one.
Prepared by 2.1 agarose gel (5g/L)
This experiment mould is 6cm × 12cm that 1.5mm is thick, and 6 hole combs, gel strength is 0.5%.
Clean two set glue mould and 6cm × 12cm comb, be placed on balancer after drying, and comb is inserted in mould
On, comb is away from mould minor face about 2.5cm.Weigh two parts of 0.15g agarose gel particle respectively, pour in two conical flasks, mark
Number " pH8.0 group ", " pH8.3 group ".The tbe buffer liquid 30ml measuring pH8.0 adds " pH8.0 group ", then measures the TBE of pH8.3
Buffer 30ml adds " pH8.3 group ".Two groups in microwave oven moderate heat heating boil glue 1min, take out, the most softly shake
Even, observing with or without undissolved granule, if having, then repeating to boil glue once.Corresponding tbe buffer liquid is added after gel is completely dissolved,
It is settled to 30ml.Gel 60min.
2.2 electrophoresis
Clean two set electrophoresis tanks, label " pH8.0 group ", " pH8.3 group ", pour corresponding tbe buffer liquid respectively into.Soft slow
Slowly, the parallel comb taking out two set glue moulds, take out gel slab, put in the electrophoresis tank of corresponding group, slowly add corresponding TBE
Buffer is to submergence gel upper surface.Take the four kinds of cell samples (i.e. the cell sample prepared in 1.3.3) prepared respectively
Appropriate (with embodiment one) from, loading in desk centrifuge point, keep electrophoresis liquid temperature constant at 10 DEG C～12 DEG C, constant voltage
100V, to bromophenol blue index strip arrive glue afterbody (altogether about 150～180min, " pH8.0 group " about needs 150min, " pH8.3 group " about
Needing 180min, the concrete time arrives glue afterbody with bromophenol blue index strip and is as the criterion).
3, separating resulting
The comparing result of difference pH when Fig. 6 .1 is agarose gel electrophoresis separation MDA-MB-231 cell LDH isozyme
(1.pH8.3、2.pH8.0).Good by two groups of equal separating effects seen from Fig. 6 .1, band is good, and the complete electrophoresis of pH8.3 group LDH4 goes out
Loading wells, it is simple to quantitative analysis, pH8.0 group LDH4 some retention and loading wells, it is unfavorable for quantitative analysis.
Claims (10)
1. lactate dehydrogenase isoenzyme electrophoresis separating method, it is characterised in that: electrophoretic buffer is tbe buffer liquid, and described TBE delays
Rushing liquid is Tris 10.8g, Na2EDTA·2H2O 0.744g, boric acid 5.5g, add distilled water and dissolve, and is settled to 1000ml and prepares.
Method the most according to claim 1, it is characterised in that: described electrophoresis separating method includes that agarose gel electrophoresis divides
From method and/or cellulose acetate membrane electrophoresis separation method.
Method the most according to claim 1, it is characterised in that: described electrophoresis separating method is that agarose gel electrophoresis separates
Method, electrophoresis temperature 10 DEG C～12 DEG C.
Method the most according to claim 1, it is characterised in that: described electrophoresis separating method is that agarose gel electrophoresis separates
Method, when described lactate dehydrogenase isoenzyme is human tumor cell's lactate dehydrogenase isoenzyme, electrophoretic buffer pH=8.3；
When described lactate dehydrogenase isoenzyme is guinea pig tissues cell lactate dehydrogenase isoenzyme, electrophoretic buffer pH=9.2；Described breast
When acidohydrogenase isozyme is rat tissue's cell lactate dehydrogenase isoenzyme, electrophoretic buffer pH=8.0.
Method the most according to claim 4, it is characterised in that: described human tumor cell is BT-549 and/or ZR-75-
30 and/or A375 and/or MDA-MB-231, described rat tissue is the rat heart and/or liver and/or kidney and/or lung and/or small intestinal
And/or skeletal muscle, described guinea pig tissues is the Cavia porcellus heart and/or liver and/or kidney and/or lung and/or intestinal and/or skeletal muscle.
Method the most according to claim 1, it is characterised in that: described electrophoresis separating method is that agarose gel electrophoresis separates
Method, 10 times of sample-loading buffers are: bromophenol blue 0.04g, 1 times of tbe buffer liquid 5ml, sucrose 10g, heat, dissolve, are cooled to room
Add 1 times of tbe buffer liquid after temperature and be settled to 10ml.
Method the most according to claim 1, it is characterised in that: described electrophoresis separating method is that agarose gel electrophoresis separates
Method, electrophoresis constant voltage 100V；Or, described electrophoresis separating method is cellulose acetate membrane electrophoresis separation method, electrophoresis constant voltage
150V。
8. the human tumor cell's lactic acid utilizing the lactate dehydrogenase isoenzyme electrophoresis separating method described in claim 1 to realize takes off
Hydrogen enzyme isoenzyme electrophoresis separating method.
Human tumor cell's lactate dehydrogenase isoenzyme electrophoresis separating method the most according to claim 8, it is characterised in that:
Described human tumor cell is BT-549 and/or ZR-75-30 and/or A375 and/or MDA-MB-231.
10.TBE buffer lactate dehydrogenase isoenzyme be separated by electrophoresis in application, described tbe buffer liquid be Tris 10.8g,
Na2EDTA·2H2O 0.744g, boric acid 5.5g, add distilled water and dissolve, and is settled to 1000ml and prepares.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107870190A (en) * | 2017-11-21 | 2018-04-03 | 中国人民解放军军事科学院军事医学研究院 | The hereditary biochemical site-tag of cerebral ischemia inbred strais meriones unguiculatus and its application |
| CN109655455A (en) * | 2019-01-31 | 2019-04-19 | 武汉大学 | A kind of kit and its application method for zooblast Species estimation |
| CN114544738A (en) * | 2022-02-15 | 2022-05-27 | 河北北方学院 | Electrophoresis buffer solution |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1431315A (en) * | 2003-01-09 | 2003-07-23 | 中国人民解放军第二军医大学 | Kit for detecting lactate dehydrogenase isoenzyme |
| CN103958694A (en) * | 2011-08-30 | 2014-07-30 | 北京沙东生物技术有限公司 | Use of detection of aspartate aminotransferase and lactate dehydrogenase in early evaluation of clinical efficacy of anti-tumor intervention |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1431315A (en) * | 2003-01-09 | 2003-07-23 | 中国人民解放军第二军医大学 | Kit for detecting lactate dehydrogenase isoenzyme |
| CN103958694A (en) * | 2011-08-30 | 2014-07-30 | 北京沙东生物技术有限公司 | Use of detection of aspartate aminotransferase and lactate dehydrogenase in early evaluation of clinical efficacy of anti-tumor intervention |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107870190A (en) * | 2017-11-21 | 2018-04-03 | 中国人民解放军军事科学院军事医学研究院 | The hereditary biochemical site-tag of cerebral ischemia inbred strais meriones unguiculatus and its application |
| CN109655455A (en) * | 2019-01-31 | 2019-04-19 | 武汉大学 | A kind of kit and its application method for zooblast Species estimation |
| CN114544738A (en) * | 2022-02-15 | 2022-05-27 | 河北北方学院 | Electrophoresis buffer solution |
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