CN102539218B - Citric acid Ethanol Method is removing the application in Human Serum Albumin - Google Patents
Citric acid Ethanol Method is removing the application in Human Serum Albumin Download PDFInfo
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- CN102539218B CN102539218B CN201010597184.6A CN201010597184A CN102539218B CN 102539218 B CN102539218 B CN 102539218B CN 201010597184 A CN201010597184 A CN 201010597184A CN 102539218 B CN102539218 B CN 102539218B
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- 238000000034 method Methods 0.000 title claims abstract description 71
- 102000008100 Human Serum Albumin Human genes 0.000 title claims abstract description 7
- 108091006905 Human Serum Albumin Proteins 0.000 title claims abstract description 7
- QJWQYOHBMUQHGZ-UHFFFAOYSA-N ethanol;2-hydroxypropane-1,2,3-tricarboxylic acid Chemical compound CCO.OC(=O)CC(O)(C(O)=O)CC(O)=O QJWQYOHBMUQHGZ-UHFFFAOYSA-N 0.000 title abstract description 12
- 239000000284 extract Substances 0.000 claims abstract description 42
- 210000002966 serum Anatomy 0.000 claims abstract description 39
- 239000006228 supernatant Substances 0.000 claims abstract description 22
- 150000002632 lipids Chemical class 0.000 claims abstract description 4
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims description 45
- 238000001556 precipitation Methods 0.000 claims description 20
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical group CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 16
- 229960004106 citric acid Drugs 0.000 claims description 13
- 239000003960 organic solvent Substances 0.000 claims description 10
- 239000004519 grease Substances 0.000 claims description 6
- 210000004369 blood Anatomy 0.000 claims description 5
- 239000008280 blood Substances 0.000 claims description 5
- 239000002904 solvent Substances 0.000 claims description 5
- 229960004543 anhydrous citric acid Drugs 0.000 claims description 3
- 239000000463 material Substances 0.000 claims description 2
- 102000004169 proteins and genes Human genes 0.000 abstract description 23
- 108090000623 proteins and genes Proteins 0.000 abstract description 23
- 238000005119 centrifugation Methods 0.000 abstract 1
- 238000005204 segregation Methods 0.000 abstract 1
- 102000009027 Albumins Human genes 0.000 description 38
- 108010088751 Albumins Proteins 0.000 description 38
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 28
- 230000000694 effects Effects 0.000 description 21
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 11
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 9
- 230000006920 protein precipitation Effects 0.000 description 8
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 6
- 239000002253 acid Substances 0.000 description 6
- BDERNNFJNOPAEC-UHFFFAOYSA-N propan-1-ol Chemical compound CCCO BDERNNFJNOPAEC-UHFFFAOYSA-N 0.000 description 6
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 4
- 102000007562 Serum Albumin Human genes 0.000 description 4
- 108010071390 Serum Albumin Proteins 0.000 description 4
- 201000010099 disease Diseases 0.000 description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 4
- 235000019441 ethanol Nutrition 0.000 description 4
- 241000196324 Embryophyta Species 0.000 description 3
- 241000283973 Oryctolagus cuniculus Species 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 239000000654 additive Substances 0.000 description 2
- 230000000996 additive effect Effects 0.000 description 2
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 239000006166 lysate Substances 0.000 description 2
- 210000002381 plasma Anatomy 0.000 description 2
- 102000004506 Blood Proteins Human genes 0.000 description 1
- 108010017384 Blood Proteins Proteins 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 238000000151 deposition Methods 0.000 description 1
- 230000008021 deposition Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 239000000469 ethanolic extract Substances 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000003317 immunochromatography Methods 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 102000035118 modified proteins Human genes 0.000 description 1
- 108091005573 modified proteins Proteins 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000000751 protein extraction Methods 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
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- Peptides Or Proteins (AREA)
Abstract
Title of the present invention is the application of citric acid Ethanol Method in removal Human Serum Albumin, relates to high-abundant protein from serum minimizing technology, is specifically related to a kind of new method that seralbumin is removed.Comprise the steps: 1) by serum centrifugal segregation lipid; 2) add haemocyanin extract, mixing is placed on-20 DEG C of refrigerators, leaves standstill 30min; 3) sample extracting solution is centrifugal; 4) centrifugation and supernatant is collected respectively.A kind of method removing Human Serum Albumin of the present invention is simple to operate, cheap, required time is short.
Description
Technical field
The present invention relates to high-abundant protein from serum minimizing technology, be specifically related to a kind of new method that seralbumin is removed.
Background technology
Hematology as a medical disciplines, along with the development of molecular biology and correlation technique achieves huge progress, diagnosing in advance and having very large application prospect in making a definite diagnosis in disease.In clinical disease diagnosis, the change of blood constituent and the generation of disease development have very important relation.In recent years, use proteomic techniques to explore blood and become a study hotspot.
Because plasma fraction is extremely complicated, therefore the key point that effective compartment analysis then becomes the research of hematoglobin protein group is carried out to it, but some high-abundance proteins but usually can cover the detection of low-abundance protein.According to having, about 22 kinds of high-abundance proteins matter occupy 98% of Total plasma protein.The albumen of all the other 1-3% then belongs to low-abundance protein, and the change of its trace all can reflect the state of body in disease marker albumen, to have very large prospect.In blood plasma, albumin occupies the over half of protein content, and its concentration is 40mg/ml.Therefore, the matter of utmost importance of hematoglobin protein group research is exactly how effectively to remove albumin.Existing method is mainly immunochromatography, but this method is expensive, and high to equipment requirement, and common laboratory is difficult to reach requirement.Comprise the method for some solvent depositions in addition, as DAC method, trichloroacetic acid (TCA) acetone method etc.But said method is not all very desirable to albuminous removal effect, DAC method is removed limited to albumin, although TCA method eliminates more albumin, some other trace protein is also removed simultaneously.This method is compared with additive method, and low toxic and environment-friendly, while at utmost removing Human Serum Albumin, decrease the loss of trace of albumin, in blood serum sample process, it has broad application prospects as far as possible.
Summary of the invention
This invention object is to provide citric acid and is removing the application in Human Serum Albumin
The chemical structural formula of citric acid involved in the present invention is
The present invention utilizes citric acid to be dissolved in ethanol solution, and for system provides suitable acid condition, albumin is efficiently removed in this sour environment.
Here is method of operating of the present invention, and its step comprises:
1) the centrifugal 10-20min of serum 10000-20000g at 4 DEG C removes lipid like material in blood.
2) the serum 20 μ l after drawing grease removal adds 40-80 μ l extract (wherein said extract is the anhydrous citric acid ethanolic solution containing volume ratio 3-5% by weight), and vortex mixes.
3) serum extract is placed in-20 DEG C of standing 10-60min.
4) by the centrifugal 10-20min of above-mentioned serum extract 10000-20000g, precipitation retains, and supernatant is transferred to new EP and manages.
5) in step 4, precipitation is placed on Rotary Evaporators and evaporates residual organic solvent, and precipitation is preserved.
6) in step 4, supernatant is placed on Rotary Evaporators and evaporates a large amount of organic solvent, and precipitation is preserved.
Remove albumin with citric acid to have the following advantages:
1) effectively high abundance albumin is removed fast;
2) cheap;
3) easy to operate;
4) agents useful for same safety and low toxicity;
Accompanying drawing explanation
Fig. 1. the chemical constitution of citric acid.
Fig. 2. variable concentrations citric acid is on the impact of albumin removal effect, figure (A): band M is standard protein SDS6H2, band 1 is former serum, and band 2-9 is (in extract, citric acid concentration is respectively 1%, 3%, 5%, 7%, 10%, 12%, 15%, 20%) electrophoretic band of the protein precipitation after extract process.Figure (B): band M is standard protein SDS6H2, and band 1 is former serum, band 2-9 are the supernatant albumin electrophoretic band after extract process.
Fig. 3. different volumes citric acid ethanol is on the impact on albumin removal effect, figure (A): band M is standard protein SDS6H2, band 1 is former serum, and band 2-9 is (citric acid ethanol extract volume is respectively 1,2,3,4,5,6,7,8 times of serum volume) electrophoretic band of the protein precipitation after extract process.Figure (B): band M is standard protein SDS6H2, and band 1 is former serum, band 2-9 are the supernatant albumin electrophoretic band after extract process.
Fig. 4. different organic solvents is on the impact of albumin removal effect, band M is standard protein SDS6H2, band 1 is former serum, and band 2-5 is (extract solvent is respectively methyl alcohol, ethanol, acetone, propyl alcohol) electrophoretic band of the protein precipitation after extract process; With the supernatant albumin electrophoretic band through extract process after of 6-9 corresponding to 2-5.
Fig. 5. different acid affects albumin removal effect, band M is standard protein SDS6H2, band 1 is former serum, with (acid is respectively phosphoric acid, acetic acid, citric acid, TCA) electrophoretic band that 2-5 is the protein precipitation after extract process, the supernatant albumin electrophoretic band through extract process after of band 6-9 corresponding to 2-5.
Fig. 6. the comparison diagram of different plant species derived sera albumin removal effect, (A) human serum albumins removal effect figure; (B) mouse serum albumin removal effect figure; (C) albumin rabbit serum matter removal effect figure.Wherein be with M to be standard protein SDS6H2, band 1 is former serum, and band 2 is the electrophoretic band of the protein precipitation after extract process; Be supernatant albumin electrophoretic band after extract process with 3.
Fig. 7. distinct methods is to mouse serum albumin removal effect figure, and (A) DAC method removes seralbumin design sketch; (B) TCA acetone method removes seralbumin design sketch; (C) citric acid Ethanol Method removes seralbumin design sketch.Wherein be with M to be standard protein SDS6H2, band 1 is former serum, and band 2 is the precipitation electrophoretic band of the albumen after extract process; Be supernatant albumin electrophoretic band after extract process with 3.
Embodiment
The present invention is set forth further below in conjunction with concrete experimental example.Should be appreciated that these experimental examples are only for illustration of the present invention, and can not the present invention be limited.
Experimental example 1 citric acid Ethanol Method removes seralbumin
Fig. 1 is the chemical structural formula of citric acid, and protein extraction procedure carries out as follows:
1) serum centrifugal (4 DEG C, 15000g, 15min) removes lipid in blood;
2) the serum 20 μ l after drawing grease removal adds 60 μ l extracts, and wherein said extract is the anhydrous citric acid ethanolic solution containing volume ratio 3% by weight, and vortex mixes;
3) serum extract is in-20 DEG C of standing 30min;
4) above-mentioned serum extract centrifugal (4 DEG C, 15000g, 15min), precipitation retains, and supernatant is transferred to new EP and manages;
5) in step 4, precipitation is placed on Rotary Evaporators and evaporates 5min, removes residual organic solvent, and precipitation is preserved.
6) in step 4, supernatant is placed on Rotary Evaporators and evaporates 25min, and after organic solvent solvent evaporate to dryness, precipitation is preserved.
Experimental example 2 different organic solvents is on the impact of albumin removal effect
According to the method different organic solvents of experimental example 1, albumin is removed.The organic solvent adopted is respectively methyl alcohol, absolute ethyl alcohol, acetone, propyl alcohol containing 3% citric acid, and result display application absolute ethyl alcohol extracts haemocyanin can reach good effect.Result is as shown in Figure 4: M is standard protein SDS6H2, and band 1 be former serum, is with 2-5 to be precipitation (extract solvent is respectively methyl alcohol, absolute ethyl alcohol, acetone, the propyl alcohol) electrophoretic band of the albumen after extract process; With the supernatant albumin electrophoretic band through extract process after of 6-9 corresponding to 2-5.
The impact of the different acid of experimental example 3 on albumin removal effect
Different acid is adopted to remove albumin according to the method for experimental example 1.The acid adopted is respectively phosphoric acid, acetic acid, citric acid, TCA, and the extraction effect of result display citric acid is best.As shown in Figure 5: band M is standard protein SDS6H2, and band 1 is former serum, band 2-5 is the electrophoretic band of the protein precipitation after extract process, the supernatant albumin electrophoretic band through extract process after of band 6-9 corresponding to 2-5.
Experimental example 4 citric acid Ethanol Method is on the impact of different plant species derived sera albumin removal effect
According to experimental example 1 to different species (people, mouse, rabbit) derived sera albumin removal effect comparison diagrams.Result shows this method all has good removal effect to albumin in different plant species derived sera.Result is as shown in Figure 6: (A) human serum albumins removal effect figure, (B) mouse serum albumin removal effect figure; (C) albumin rabbit serum removal effect figure.Wherein M is standard protein SDS6H2, and band 1 is former serum, and band 2 is the electrophoretic band of the protein precipitation after extract process; Be supernatant albumin electrophoretic band after extract process with 3.
Experimental example 5 citric acid Ethanol Method removes seralbumin and additive method compares
Distinct methods is adopted to remove mouse serum albumin, as shown in Figure 7: (A) DAC method; (B) TCA acetone method; (C) citric acid Ethanol Method.Wherein M is standard protein SDS6H2, and band 1 is former serum, and band 2 is the precipitation electrophoretic band after extract process; Be supernatant albumin electrophoretic band after extract process with 3.The effect that result display DAC method is removed albumin is poor, and TCA acetone method albuminously also eliminates some other low-abundance protein removing simultaneously.And citric acid Ethanol Method mainly eliminates albumin, effectively remain low-abundance protein.Wherein citric acid Ethanol Method is carried out according to experimental example 1, and DAC method and TCA acetone method carry out as follows:
DAC method
[1]:
1) in 200 μ l grease removal serum, NaCl is added to final concentration 0.1M, 4 DEG C of vibration 60min.
2) add in ice ethanol to step 1 solution to ethanol final concentration be 42%, 4 DEG C place 60min.
3) centrifugal (4 DEG C, 16000g, 45min) are precipitated 1 and supernatant.
4) supernatant adds sodium acetate (pH4.0,0.8M) and adjusts pH to 5.7, places 60min.
5) centrifugal (4 DEG C, 16000g, 45min) are precipitated 2 and supernatant.
6) precipitate 1 and precipitation 2 mixing, add lysate and dissolve.
TCA acetone method
[2]:
1) add 120 μ lTCA acetone solns (wherein said extract is the acetone soln containing volume ratio 10%TCA by weight) in 40 μ l grease removal serum, after mixing, place 90min for-20 DEG C.
2) centrifugal (4 DEG C, 15000g, 20min) are precipitated and supernatant.
3) in precipitation, add 1ml acetone rinsing precipitation, after placing 15min, centrifugal (4 DEG C, 15000g, 20min) are precipitated 1.
4) add 1ml acetone to supernatant, after placing 15min, centrifugal (4 DEG C, 15000g, 20min) are precipitated 2.
5) precipitation 1, precipitation 2 respectively add lysates respectively and dissolve.
List of references:
1.Effective removal of albumin from serum，Proteomics 2005，5，3831-3835.
2.A modified protein precipitation procedure for efficient removal of albumin from serum，Electrophoresis2005，26，2117-2127.
Claims (6)
1. remove a method for Human Serum Albumin, comprise the steps:
1) the centrifugal 10-20min of serum 10000-20000g at 4 DEG C removes lipid like material in blood;
2) the serum 20 μ l after drawing grease removal adds 40-80 μ l extract, and wherein said extract is the anhydrous citric acid ethanolic solution containing volume ratio 3-5% by weight, and vortex mixes;
3) serum extract is placed in-20 DEG C of standing 10-60min;
4) the centrifugal 10-20min of extract 10000-20000g, precipitation retains, and supernatant is transferred to new EP and manages;
5) step 4) in precipitation be placed on Rotary Evaporators and evaporate residual organic solvent, precipitation is preserved;
6) step 4) in supernatant be placed in evaporate to dryness organic solvent on Rotary Evaporators, precipitation preserve.
2. the method for claim 1, is characterized in that, step 1) serum centrifugal 15min of 15000g at 4 DEG C.
3. the method for claim 1, is characterized in that, step 2) to draw the serum 20 μ l after grease removal and add 60 μ l extracts, vortex mixes.
4. as claim 1-3 arbitrary as described in method, it is characterized in that, step 2) concentration of citric acid is 3% in extract, solvent is absolute ethyl alcohol.
5. the method for claim 1, is characterized in that, step 3) serum extract is placed in-20 DEG C of standing 30min.
6. the method for claim 1, is characterized in that, step 4) the centrifugal 15min of extract 15000g.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106153420B (en) * | 2016-07-29 | 2020-04-17 | 季伙燕 | Pretreatment process for quantitatively detecting serum low-abundance protein by isotope dilution mass spectrometry |
| CN113176133B (en) * | 2021-03-15 | 2024-02-13 | 广州邦德盛生物科技有限公司 | Method for separating protein and lipid in blood plasma or serum and matrix serum |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4093612A (en) * | 1975-08-04 | 1978-06-06 | Research Corporation | Selective removal of albumin from blood fluids and compositions therefore |
| JPS6357597A (en) * | 1986-08-28 | 1988-03-12 | Ube Ind Ltd | Removal or recovery of serum albumin |
| EP1141021B1 (en) * | 1998-12-22 | 2004-09-08 | Amersham Biosciences AB | Removal/purification of serum albumins |
| CN101165466A (en) * | 2006-10-19 | 2008-04-23 | 陕西西大北美基因股份有限公司 | Method for removing high abundance protein using gold magnetic particle |
| CN101793865A (en) * | 2010-03-29 | 2010-08-04 | 内蒙古蒙牛乳业（集团）股份有限公司 | Method for detecting content of human seralbumin in human milk |
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2010
- 2010-12-10 CN CN201010597184.6A patent/CN102539218B/en not_active Expired - Fee Related
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4093612A (en) * | 1975-08-04 | 1978-06-06 | Research Corporation | Selective removal of albumin from blood fluids and compositions therefore |
| JPS6357597A (en) * | 1986-08-28 | 1988-03-12 | Ube Ind Ltd | Removal or recovery of serum albumin |
| EP1141021B1 (en) * | 1998-12-22 | 2004-09-08 | Amersham Biosciences AB | Removal/purification of serum albumins |
| CN101165466A (en) * | 2006-10-19 | 2008-04-23 | 陕西西大北美基因股份有限公司 | Method for removing high abundance protein using gold magnetic particle |
| CN101793865A (en) * | 2010-03-29 | 2010-08-04 | 内蒙古蒙牛乳业（集团）股份有限公司 | Method for detecting content of human seralbumin in human milk |
Non-Patent Citations (4)
| Title |
|---|
| A modified protein precipitation procedure for efficient removal of albumin from serum;Yi-Yun Chen et al;《Electrophoresis》;20051231;第26卷;第2117-2127页 * |
| Comparison of protein precipitation methods for sample preparation prior to proteomic analysis;Lei Jiang et al;《Journal of Chromatography A》;20041231;第1023卷;第317-320页 * |
| Effective removal of albumin from serum;David A. Colantonio et al;《Proteomics》;20051231;第5卷;第3831–3835页 * |
| 疏水层析结合冷乙醇沉淀纯化人血清白蛋白;杨海荣等;《生物工程学报》;20041130;第20卷(第6期);第1.2.3、1.2.4、2.1及2.2小节 * |
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