CN108727464A - A kind of new Protein Extraction Reagent and method - Google Patents
A kind of new Protein Extraction Reagent and method Download PDFInfo
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- CN108727464A CN108727464A CN201810577627.1A CN201810577627A CN108727464A CN 108727464 A CN108727464 A CN 108727464A CN 201810577627 A CN201810577627 A CN 201810577627A CN 108727464 A CN108727464 A CN 108727464A
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- cell
- protein
- protein extraction
- tris
- extraction reagent
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/145—Extraction; Separation; Purification by extraction or solubilisation
Abstract
The present invention provides a kind of Protein Extraction Reagents, include following components per 10ml aqueous solutions：14.5 ~ 15.5mg of Tris, 74 ~ 75mg of glycine, SDS 202-208mg, 10 ~ 12mg of bromjophenol blue, 0.5 ~ 0.6ml of Tris-HCl, 0.8 ~ 1.2ml of glycerine, beta -mercaptoethanol 0.4-0.6ml.The present invention not only solves the test problems of few cells, improves the detection sensitivity of Western blot.
Description
Technical field
The invention belongs to biotechnologies, are related to protein extraction.
Background technology
Western blot (western blot) are the detection most important experimental method of albumen in scientific research industry, as long as being related to
To Protein Detection, first choice must be Western blot.Western blot are molecular biology, biochemistry and immune genetic
Common a kind of experimental method in.Its basic principle is by specific antibody to the processed cell of gel electrophoresis or biology
Histone sample is coloured.Position by analyzing coloring obtains specific protein in the cell analyzed with color depth
Or tissue in expression information.It is also most important protein detection technology that it, which is most common,.But carrying out Western
Before blot, it is necessary to extract cell or the total protein of biological tissue.It is detection pair that Western blot, which are with the total protein of extraction,
An experimental technique for elephant.
In general, the total protein of biological tissue is relatively good extraction, because tissue mass is more than enough, total egg of extraction
More than enough, the enough height of total protein concentration are measured in vain.But the total protein extraction for cultivating cell is not just so easy.Because of Western
Blot requires the total protein concentration and concentration of extraction.Itself protein content of the cell of culture is far fewer than the albumen in tissue
Amount, even if plus traditional lysate can propose total protein, but albumen concentration is usually not enough to be detected by Western blot
Come.Want to carry high protein concentration when this, it is possible to reduce the dosage of lysate, but after reducing lysate, total egg of extraction
White amount can reduce again, and concentration reaches and protein content deficiency can not carry out Western blot experiments.Therefore, in order to ensure to cultivate
The protein content and concentration of cell.Just only increase the quantity of culture cell.In traditional means of experiment, best situation also at least needs
The protein content in 2 holes in 6 orifice plates is wanted just to be enough to be Western blot.
In most of practical operation, the protein content in 3 to 4 holes in 6 orifice plates can be generally used.The increase of this cell concentration is most
Our experiment material cost and cost of labor can directly be increased.Secondly, such case can consume more resources, generate more
Rubbish and harmful product, it is extremely unfavorable to environmental protection.Furthermore since the cell concentration of needs is big, inevitably increase when being processed to cell
Add operation error, influences experimental result.It is finally and most important, for a variety of cells especially primary cell, rare thin
Born of the same parents, newly cultivate cell, precious cell, non-proliferative cell etc. we in experiment, cannot get at all required for tradition extraction albumen
Protein content, Western blot can not be carried out.So we are badly in need of a kind of new high efficiency extraction total protein method to solve this
A little problems.
Invention content
The purpose of the present invention is to provide a kind of new Protein Extraction Reagent, this detection for not only solving few cells is asked
Topic, also improves the detection sensitivity of Western blot.
The purpose of the present invention is what is realized by following measures：
A kind of Protein Extraction Reagent includes following components per 10ml aqueous solutions：Tris (trishydroxymethylaminomethane)
14.5~15.5mg, 74~75mg of glycine, SDS 202-208mg, 10~12mg of bromjophenol blue, 0.5~0.6ml of Tris-HCl,
0.8~1.2ml of glycerine, beta -mercaptoethanol 0.4-0.6ml.
Preferably, the optimum proportioning of each component is in above-mentioned Protein Extraction Reagent：Containing with the following group in per 10ml aqueous solutions
Point：Tris 15mg, glycine 75mg, SDS 205mg, the Tris-HCl0.5ml of bromjophenol blue 10mg, pH6.8, glycerine 1ml, β-
Mercaptoethanol 0.5ml.
Above-mentioned Protein Extraction Reagent dosage is per 1x106400 μ of μ L~600 L are added in cell；Or 24 orifice plate add 200 per hole
The μ of μ L~300 L.
Another object of the present invention is to provide the methods for extracting albumen using mentioned reagent.
A kind of protein extracting method, includes the following steps：
(1) cell is collected, according to every 1x10 after cleaning6Protein Extraction Reagent described in 400 μ of μ L~600 L is added in cell；
(2) static 30 seconds to 2 minutes；
(3) boiling 10~20 minutes makes albuminous degeneration, cooling.
Specifically, the application method of above-mentioned Protein Extraction Reagent, includes the following steps：
(1) Protein Extraction Reagent is prepared：It is proportionally added into Tris, glycine, SDS, bromjophenol blue, Tris-HCI, glycerine, β-
Mercaptoethanol, water；
(2) for attached cell：Culture solution is removed, is washed with buffer solution, 200 μ of μ L~300 L are added per hole according to 24 orifice plates
The reagent；Mixing is gently blown and beaten, extracting solution and cell is made to come into full contact with；For suspension cell：Cell is collected by centrifugation, breaks up thin
Born of the same parents are added the 200 μ L of μ L~300 per hole cell according to 24 orifice plates, gently blow and beat mixing, extracting solution and cell is made to come into full contact with；
(3) 100 DEG C or boiling water bath heat 15 minutes, with abundant albuminate；It is stored in -20 DEG C after albuminous degeneration, waits for
Western loadings are used.SDS-PAGE sets 20 microlitres per hole applied sample amount.Sample can save 1 year.
According to 24 orifice plate of above step 10 western can be per hole.The new method that converts only needs conventional method eight
/ 10th protein content.
Advantageous effect
1. tradition extraction albumen is the biggest problems are that a large amount of cells will be cultivated, but not all cell can be carried out greatly
Amount culture, effective total protein concentration cannot be extracted for micro cell conventional method.But the present invention only needs conventional method
Eightieth protein content, this not only solves the test problems of few cells, also improves Western blot
Detection sensitivity.
2. the present invention is not only helpful to being unable to the cell of mass propgation, the sensitivity of Protein Detection is improved；To normal
The cell protein detection of culture is also helpful.Firstly, since cell dosage is seldom, a detection group only needs 24 orifice plate, one hole
Cell, it is possible to reduce culture to cell, saved time and money cost；Secondly, the cell in 24 one hole of orifice plate
Amount plus 200 microlitres of new albumen sample solutions, are 10 Western blot enough, ensure that repetition detects dosage, improve
The detection accuracy of Western blot.
3. step of the present invention is simple to operation, it is only necessary to a few minutes i.e. extractable completion.This invention simplifies experimental procedure,
So that experimental implementation error rate reduces.Same sample process, the present invention only need one or two minute in protein cleavage link, and old
Method needs to operate on ice 30 minutes or more, and the time greatly reduces.Due to the time reduction also there is no need to it is whole on ice
Operation, simplifies Preparatory work of experiment.
4. in tradition extracts albumen, some operators can carry out Concentration Testing to albumen after extracting albumen, further according to dense
Degree adjusts cracking liquid measure again so that the loading total protein with batch difference group keeps same concentration, such Western blot to run out of
Internal reference β-actin between the different samples come are theoretically consistent, and the difference of destination protein seems more very clear, it
Benefit is conveniently intuitively to see figure judging result, but quantitative analysis results are with the no necessarily relationship of this operation.And in practical behaviour
In work, internal reference still can not possibly be accomplished equally, usually to run out irregular.For this difference, inventor provides two ginsengs
Examine solution.The first scheme is directly surveyed with OD-1000 OD-2000 spectrophotometers after new method extracts albumen
Albumen concentration, so that it is determined that applied sample amount, unified internal reference.But inventor's more suggested design two.Scheme two directly subtracts albumen concentration
This step is detected, operating procedure and time are reduced, alternatively maintains internal reference unified.It comprises the concrete steps that and is spread in cell culture
When plate, cell count is carried out so that it is the same per the cell number under the kind of hole, then being just to determine under kind per the total protein concentration of hole cell
Determine to be the same, the internal reference run out i.e. much the same.If the internal reference that albumen is run out is widely different, can adjust
Whole applied sample amount so that internal reference is neat so that result is convenient for observation.
5. protein extraction of the present invention is complete, from 15KD to 22KD for, complete extraction and can all detected, completely can be with
Instead of conventional reagents and method.
6. the present invention is of less demanding to cell state.Whether dosing or physical mechanical processing efficiently can be carried completely
Take albumen.It in actual experiment, alternates betwwen good and bad by various processing cell states, proliferative capacity is much begun to decline, or even thin
Born of the same parents senesce apoptosis aggravation, but no matter the cell present invention of which kind of state can efficient complete extraction.
7. the present invention is suitable for various cells.It can either be extracted for human archeocyte or mouse source cell.Even if
It is primary cell, culture is very difficult, can also complete extraction albumen.
8. the present invention is more efficient.Conventional method needs initial cell amount big；And the present invention needs cell concentration low.Tradition side
Method is that albumen is stripped out from cell, and extraction is imperfect；The present invention is not need protein isolate matter and cell other compositions,
Obtained protein is more complete, and experimental result is more acurrate effectively.Traditional method for extracting amount is not high；The present invention extracts full amount and fills
Foot.
Description of the drawings
Fig. 1 present invention stimulates human umbilical cord mesenchymal stem cells for IFN-γ
Fig. 2 present invention acts on Marrow Mesenchymal Stem Cells for LIPUS and 2-APB
Fig. 3 β-actin compare new out-of-date methods leach protein difference on effect
Specific implementation mode
The present invention is specifically described below by embodiment, it is necessary to which indicated herein is that following embodiment is only used
In invention is further explained, it should not be understood as limiting the scope of the invention, field technology personnel can be with
Some nonessential modifications and adaptations are made to the present invention according to aforementioned present invention content.
Embodiment 1
A kind of Protein Extraction Reagent weighs 14.5~15.5mg of Tris, 74~75mg of glycine, SDS 205mg, bromine phenol
10~12mg of orchid, Tris-HCI (pH6.8) 0.5~0.6ml, 0.8~1.2ml of glycerine, beta -mercaptoethanol 0.5ml, be added go from
Sub- water is settled to 10ml.- 20 DEG C can save 1 year.
1. the Protein Extraction Reagent stimulates human umbilical cord mesenchymal stem cells for IFN-γ
(1) primary human umbilical cord mesenchymal stem cells are detached.Cultivate the primary human umbilical cord mesenchymal stem cells of the third generation.
1x10 is added in (2) 24 orifice plates per hole5A human umbilical cord mesenchymal stem cells add various concentration IFN- after adherent
γ (a kind of drug can stimulate the related protein factor of aim cell secretion), IFN-γ concentration be respectively 0ng/ml, 20ng/ml,
5ng/ml0,100ng/ml.Culture 48 as a child prepared to collect cell protein.
(3) culture medium is removed, buffer solution for cleaning 3 times adds 200 μ L Protein Extraction Reagents per hole according to 24 orifice plates, gently blows
Mixing is beaten, static 1 minute, liquid was packed into EP pipes.It boils 15 minutes, makes albuminous degeneration.- 20 DEG C are put after cooling to freeze.
(4) after extracting albumen, the destination protein developed by molecule in Western blot detection human umbilical cord mesenchymal stem cells
Amount.Have detected protein β-actin, LC3, P62, Beclin.Every group takes 20uL albumen, and electric robin is by Protein transfer to PVDF
On film, TBST is washed 5 times, each 3min；5% skimmed milk power room temperature closes 1h, and β-actin antibody, LC3, P62, Beclin is added
Antibody (1：1000 dilutions), 4 DEG C of overnight incubations, TBST is washed 5 times, each 3min, adds secondary antibody, and TBST is washed after being incubated at room temperature 4h
It washs 5 times, each 3min, chemoluminescence method colour developing.
Experimental result is shown in Fig. 1.It is 15KD, P62 albumen that wherein β-actin molecular weight of albumen, which is 45KD, LC3 molecular weight of albumen,
Molecular weight is that 62KD, Beclin molecular weight of albumen are 60KD.The molecular weight of protein is generally 10 between 250KD, LC3 molecules
Amount 15KD can represent the extractability of small molecular weight protein, other albumen can represent the protein extraction energy of intermediate molecular weight
Power.(CST companies of the anti-LC3 antibody U.S., abcam companies of the anti-Beclin1 antibody U.S., the anti-P62 antibody U.S.
Sigma companies, sigma companies of the U.S. β-actin)
2. the Protein Extraction Reagent acts on Marrow Mesenchymal Stem Cells for LIPUS
(1) Primary mouse mesenchymal stem cell is detached.Cultivate the third generation.
1x10 is added in (2) 24 orifice plates per hole5A Marrow Mesenchymal Stem Cells use low-strength focusing ultrasonic after adherent
(LIPUS) cell is handled, culture 48 as a child prepared to collect cell protein.
(3) culture medium is removed, buffer solution for cleaning 3 times adds 200 μ L extracting solutions per hole according to 24 orifice plates, gently blows and beats mixing,
Static 1 minute, liquid was packed into EP pipes.It boils 15 minutes, makes albuminous degeneration.- 20 DEG C are put after cooling to freeze.
(4) after extracting albumen, the destination protein molecule table in Western blot detection Marrow Mesenchymal Stem Cells
Up to amount.Have detected protein G APDH, TRPM7.Every group takes 20uL albumen, and electric robin is by Protein transfer to pvdf membrane, TBST
It washes 5 times, each 3min；5% skimmed milk power room temperature closes 1h, and GAPDH, TRPM7 antibody (1: 1000 dilution), 4 DEG C of incubations are added
Overnight, TBST is washed 5 times, each 3min, adds secondary antibody, and TBST is washed 5 times, each 3min after being incubated at room temperature 4h, chemiluminescence
Method develops the color.
Experimental result is shown in Fig. 2.Fig. 2 is that LIPUS acts on Marrow Mesenchymal Stem Cells, the Trpm7 albumen point of detection
It is 36KD that son amount, which is 220KD, GAPDH molecular weight of albumen,.The molecular weight of protein is generally 10 between 250KD, TRPM7 molecules
Amount 220KD can represent the extractability of macromolecular weight protein.(U.S. GAPDH santa cruz, TRPM7 Britain Abcam)
3. the Protein Extraction Reagent compares new out-of-date methods leach protein difference by β-actin
(1) primary human umbilical cord mesenchymal stem cells are detached.Cultivate the primary human umbilical cord mesenchymal stem cells of the third generation.
(2) 8x10 is added in T25 culture bottles5A human umbilical cord mesenchymal stem cells；1x10 is added in 24 orifice plates per hole5It is personal
Umbilical cord mesenchymal stem cells.Culture 48 as a child prepared to collect cell protein.
(3) the method for the present invention：Culture medium is removed, buffer solution for cleaning 3 times adds 200 μ L embodiments, 1 institute according to 24 orifice plates per hole
Protein Extraction Reagent is stated, gently blows and beats mixing, static 1 minute, liquid was packed into EP pipes.
Conventional method：The total protein in a T25 culture bottle is extracted, operation is as follows：Egg is prevented because extraction time is long
White degradation whole process operates on ice.Melt RIPA lysates, mixing.The lysate for taking appropriate amount adds using in first several minutes
Enter PMSF (protease inhibitors), it is 1mM to make the ultimate density of PMSF.Culture solution is removed, is washed three times with buffer solution.According to 6 holes
Lysate, i.e. every bottle of T25 plus 100 microlitres of lysates is added in the ratio that 40 microlitres of lysates are added per hole for plate.With rifle piping and druming it is several under,
Lysate and cell is set to come into full contact with.Mixing concussion is put in be cracked on ice, shakes mixing repeatedly every 10min, totally 3 times.Fully
After cracking, 10000g is centrifuged 5 minutes, takes 80 microlitres of supernatant.Dissolve SDS-PAGE sample-loading buffers (5X).80 microlitres of lysates add
Enter 20 microlitres of SDS-PAGE sample-loading buffers (5X).Wherein, required whole reagent RIPA lysates, PMSF, SDS-PAGE loading
Buffer solution (5X) is all commercially available commodity.
(4) it boils 15 minutes, makes albuminous degeneration.- 20 DEG C are put after cooling to freeze.
(5) after extracting albumen, Western blot detect the β-actin in human umbilical cord mesenchymal stem cells, and applied sample amount is equal
It is 20 microlitres.
Experimental result is shown in Fig. 3 by WB.(1) conventional method is added 40 microlitres per hole according to above step optimal cases 6 orifice plates and splits
Liquid is solved, 50 microlitres of samples can be obtained, 20 microlitres of applied sample amount, i.e. 2 hole of 6 orifice plates can at most be 5 western.And 24 holes of the invention
Plate can be 10 western per hole.The present invention that converts only needs the eightieth protein content of conventional method.(2) Fig. 3 A
It is the comparison of the β-actin of the albumen in 24 one hole of orifice plate of albumen and new method extraction that traditional approach extracts a T25.Tradition
It is 10: 1 that method and the method for the present invention, which collect the comparison of cell concentration,.Fig. 3 B are the gray analysis values of β-actin contained by WB bands.Its
Data illustrate that the method for the present invention only has 1/10th of conventional method, the present invention to extract the β-of protein in cell concentration
Actin expression quantity is the three times of conventional method or more.
Claims (5)
1. a kind of Protein Extraction Reagent includes following components per 10ml aqueous solutions：Tris14.5 ~ 15.5mg, glycine 74 ~
75mg, SDS 202-208mg, 10 ~ 12mg of bromjophenol blue, 0.5 ~ 0.6ml of Tris-HCl, 0.8 ~ 1.2ml of glycerine, beta -mercaptoethanol
0.4-0.6ml。
2. Protein Extraction Reagent as described in claim 1 contains Tris 15mg, glycine 75mg, SDS per 10ml aqueous solutions
205mg, Tris-HCl 0.5ml of bromjophenol blue 10mg, pH6.8, glycerine 1ml, beta -mercaptoethanol 0.5ml.
3. Protein Extraction Reagent as claimed in claim 1 or 2, dosage is per 1x106Cell is added described in 400 μ of μ L ~ 600 L and tries
Agent；Or 24 orifice plate add reagent described in 200 μ of μ L ~ 300 L per hole.
4. using the method for the Protein Extraction Reagent extraction albumen of claim 1,2 or 3, include the following steps：
（1）Cell is collected, according to every 1x10 after cleaning6Protein Extraction Reagent described in 400 μ of μ L ~ 600 L is added in cell；
（2）Static 30 seconds to 2 minutes；
（3）Boiling 10 ~ 20 minutes makes albuminous degeneration, cooling.
5. the method for extraction albumen as claimed in claim 4, includes the following steps：
（1）Prepare Protein Extraction Reagent：It is proportionally added into Tris, glycine, SDS, bromjophenol blue, Tris-HCl, glycerine, β-sulfydryl
Ethyl alcohol, water；
（2）For attached cell：Culture solution is removed, is washed with buffer solution, is added per hole according to 24 orifice plates and is tried described in 200 μ of μ L ~ 300 L
Agent；Mixing is gently blown and beaten, extracting solution and cell is made to come into full contact with；
For suspension cell：Cell is collected by centrifugation, breaks up cell, the 200 μ L of μ L ~ 300 are added per hole cell according to 24 orifice plates, gently
Mixing is blown and beaten, extracting solution and cell is made to come into full contact with；
（3）100 DEG C or boiling water bath heat 15 minutes, with abundant albuminate；Be stored in after albuminous degeneration -20 DEG C it is spare.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1131439A (en) * | 1993-08-17 | 1996-09-18 | 莫根国际公司 | Chitinase DNA coding thereof and plants containing same |
| CN101754976A (en) * | 2007-07-24 | 2010-06-23 | 保罗·普里米耶罗 | Complexes of Grp94 with human immunoglobulin G |
| US20130219570A1 (en) * | 2009-04-17 | 2013-08-22 | Dow Agrosciences Llc | DIG-3 INSECTICIDAL Cry TOXINS |
-
2018
- 2018-06-06 CN CN201810577627.1A patent/CN108727464A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1131439A (en) * | 1993-08-17 | 1996-09-18 | 莫根国际公司 | Chitinase DNA coding thereof and plants containing same |
| CN101754976A (en) * | 2007-07-24 | 2010-06-23 | 保罗·普里米耶罗 | Complexes of Grp94 with human immunoglobulin G |
| US20130219570A1 (en) * | 2009-04-17 | 2013-08-22 | Dow Agrosciences Llc | DIG-3 INSECTICIDAL Cry TOXINS |
Non-Patent Citations (4)
| Title |
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| H. SCHIECHL: "Isolation, chemical characterization, and immunohistochemical localization of a protein from the basolateral plasma membrane of the rat intestinal absorptive cell", 《Z NATURFORSCH C J BIOSCI》 * |
| 季一兵: "《中药毛细管电泳分析技术与应用》", 30 June 2009, 中国医药科技出版社 * |
| 钱士匀: "《分子诊断学实验指导》", 31 December 2006, 高等教育出版社 * |
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Application publication date: 20181102 |