CN106324249A - Sample pretreatment method of secretory proteins in culture medium with serum - Google Patents
Sample pretreatment method of secretory proteins in culture medium with serum Download PDFInfo
- Publication number
- CN106324249A CN106324249A CN201510387358.9A CN201510387358A CN106324249A CN 106324249 A CN106324249 A CN 106324249A CN 201510387358 A CN201510387358 A CN 201510387358A CN 106324249 A CN106324249 A CN 106324249A
- Authority
- CN
- China
- Prior art keywords
- albumen
- cell
- protein
- serum
- culture fluid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
- G01N33/6848—Methods of protein analysis involving mass spectrometry
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Bioinformatics & Computational Biology (AREA)
- Chemical & Material Sciences (AREA)
- Urology & Nephrology (AREA)
- Immunology (AREA)
- Biomedical Technology (AREA)
- Hematology (AREA)
- Cell Biology (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Microbiology (AREA)
- Biophysics (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Food Science & Technology (AREA)
- Biotechnology (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
Abstract
The invention belongs to the field of biochemical analysis, and relates to a sample pretreatment method of secretory proteins in culture medium with serum. High abundance protein removing technology and protein fractionation technology are used for enriching secretory proteins, and furthermore mass spectrometry is used for identification. The method has the advantages of simple operation, time saving and high efficiency. The method is used for identification of quantity of secretory proteins in culture medium with serum, and the method compares favourably with serum-free culture technology.
Description
Technical field
The present invention relates to biochemical analysis field, specifically, be by high-abundance proteins removal technology and
Albumen classification technique enrichment secretory protein, and it is carried out mass spectral analysis qualification.
Background technology
Secretory protein refers to the class protein being secreted into extracellular function.Secretory protein includes
Many somatomedin and cytokine, wide participation cellular signal transduction, immunne response, the increasing of cell
Grow, the vital movement that numerous organisms such as regulation and control of differentiation and apoptosis are important.Secretory protein group deep
Enter to study to contribute to us and find the biomarker the most relevant to morbid state, and dividing
In sub-level, the generation to disease develops offer theoretical foundation.
It is the grinding of proteomics in recent years for the research of the secretory protein group of different excretory systems
Study carefully focus, especially for cancerous cell dissimilar, different phase, there is highly important research meaning
Justice.Traditional secretory protein group research strategy overwhelming majority uses the condition of culture of serum-free, to keep away
Exempt from the very big ambient interferences that secretory protein itself is caused by high abundance plasma proteins group.But, for
For cell, this Hunger stress of serum-free culture can change the secretion behavior of cell self, the most sharp
Living cells apoptosis pathway.So, the secretory protein group reflection of qualification is under cell abnormal condition
Secretory protein group.In order to solve this difficult problem, Eichelbaum collaboration person develops one
Azido compound metabolic marker the strategy by click chemistry enrichment secretory protein, from blood serum medium
In identify 325 proper secretory proteins, secretory protein selectivity reaches 71%
(Eichelbaum et al.,Nat.Biotechnl.30,2012).But, the process employs nitrine chemical combination
Thing metabolic marker with substitute methionine strategy, although the method broken away from serum starvation yoke but
But it is the state of a kind of methionine hunger simultaneously for cell, the propagation of the growth of cell can be produced
Raw unpredictable impact, it is impossible to reflect the cell under normal cultivation conditions.
Based on this, we have developed the secretory protein sample pretreatment of a kind of direct process serum free culture system liquid
Method.Only by culture fluid being carried out the removal of high-abundance proteins and the classification of albumen, just can reach
The purpose of Large scale identification secretory protein group, does not affect the normal physiological status of cell simultaneously.
Summary of the invention
For achieving the above object, the technical solution used in the present invention is:
Take the cell culture fluid containing serum, by centrifugation, filtration treatment, then utilize high-abundance proteins successively
Removal technology and albumen classification technique process, and it is carried out enzymolysis.
The described cell culture fluid containing serum is containing 1%-30% hyclone and for cell cultivation
SILAC culture fluid；Above-mentioned SILAC culture fluid is containing isotope-labeled lysine, and arginine is bright
Propylhomoserin, isoleucine, methionine, the cell training of the combination of one or two or more kinds in agedoite
Nutrient solution.
Described centrifugal, filtration treatment, is specially the centrifugal force first with 300-700g and removes cell, from
The heart time is 1-60 minute；Take supernatant and remove cell debris with the centrifugal force of 2000-5000g again, from
The heart time is 1-60 minute, takes the membrane filtration of supernatant 0.1-1.0 μm, takes filtrate.
It is antibody column removal technology or the one of protein balancing technique that described high-abundance proteins removes technology
Or two kinds；It is to utilize several immobilized antibody to its specific high abundance egg that antibody column removes technology
Realize the purpose that specificity capture reaches to remove in vain；Protein balancing technique is to utilize specific material pair
Protein realizes equilibrium, to reduce high-abundance proteins content, improves low-abundance protein content.
Described albumen classification technique is albumen isoelectric point, IP classification technique or the one of molecular weight of albumen classification technique
Plant or two kinds.Albumen isoelectric point, IP classification technique is that it is carried out by the isoelectric point, IP character utilizing protein different
Separate；Molecular weight of albumen classification technique is that it is separated by the character utilizing protein different molecular weight.
Described enzymolysis is that albumen in the one of trypsin, serine protease or protein incision enzyme or is appointed
Anticipate under the effect of two kinds, be peptide fragment by Proteolytic enzyme；The addition of enzyme be 1:1-1:200 (enzyme: albumen,
Mass ratio)；Enzymatic hydrolysis condition is 20-50 degree Celsius of lower enzymolysis 1-48 hour.
Particularly as follows: first collect cell culture fluid, then it is carried out the slow-revving centrifugal place of the first step
Reason (300-700g) is to remove the cell of residual, then carries out the centrifugal treating of second step higher rotation speed
(2000-5000g) to remove the cell debris of residual.Then, one is entered with the filter membrane of 0.1-1.0 μm
Step removes the cell in culture fluid and cell debris.Then, utilize high-abundance proteins removal technology (anti-
Scapus removes technology or protein balancing technique) remove the high-abundance proteins in cell culture fluid, and profit
With albumen classification technique (albumen isoelectric point, IP classification technique or molecular weight of albumen classification technique) to process after
Culture fluid carry out the classification of albumen.Then, to the albumen of different fractions respectively at trypsin, silk
Enzyme is carried out under the effect of any one or more than one the combination of serine protease or protein incision enzyme
Solve.Finally, to the peptide fragment flight time class mass spectrum obtained, ion trap class mass spectrum and track trap class matter
One or more combination in spectrum carries out mass spectral analysis.
Compared with tradition serum-free culture enrichment secretory protein, the invention have the advantages that
(1) present invention can overcome the serum starvation impact on cell, maintains cell normal physiology shape
State；
(2) present invention can reduce the broken to reduce the interference of cell nuclear protein of cell；
(3) legitimate reading of cell secretory protein under the conditions of the present invention can reflect normally.
Accompanying drawing explanation
Fig. 1 is the flow chart of the present invention.
Detailed description of the invention
The present invention presented below two kinds enrichment and the detailed description of the invention of qualification secretory protein.
Embodiment 1
Utilize ProteoMiner equalizer material and Gelfree 8100 albumen classification instrument that SILAC is marked
The HeLa cell culture fluid of note realizes enrichment and the qualification of secretory protein.
First collecting the culture fluid of the HeLa cell of SILAC labelling (K6, R10), 500g is centrifuged
To remove the cell of residual, take supernatant and carry out the centrifugal cell debris remained with removing of 3000g again.Connect
, the filter membrane taking supernatant 0.22 μm carries out filtering to remove further cell debris, and collection flows through
Component.Then, ProteoMiner equalizer processes is utilized to flow through component to remove high abundance egg therein
White Sync enrichment low-abundance protein, albumen is carried out by the Gelfree 8100 of recycling Expedeon company
The classification of molecular weight, and collect each fraction.Then, the albumen in each fraction adds 100 μ L 0.1M
56 degrees Celsius of degeneration reductase 12s hour of dithiothreitol, DTT, then lucifuge adds the iodacetyl of 100 μ L0.2M
Amine room temperature alkylated reaction half an hour, it is subsequently added into the ammonium hydrogen carbonate of 1mL 50mM, adds pancreas egg
White enzyme (1:50, enzyme: albumen, mass ratio) 37 degrees Celsius is hatched 16 hours, then with desalination post pair
Sample desalination, finally carries out nano-ESI-LC-MS/MS mass spectral analysis.
ProteoMiner equalizer processes condition: first the albumen of 200 μ L60mg/ml is flowed through component
With 100mg ProteoMiner equalizer material incubated at room under the conditions of 10mM HEPESpH=7.0
2h.After having hatched, flush three times with 10mM HEPES, with 300 μ L eluents (4%SDS,
25mM DTT) under the conditions of 95 DEG C, hatch 15min, then carry out 500g and be centrifuged eluted product.
Eluent is the culture fluid after eliminating high-abundance proteins.
Gelfree 8100 albumen staged care condition: according to the operation instruction of instrument, takes 20 μ L
SampleBuffer and 10 μ L Running Buffer (producer provides by instrument), two sulfur of 8 μ L 1M
Threitol and the 112 pending sample mix of μ L are placed in the sample cell of instrument, the program recommended according to instrument
Voltage arranges and sample is carried out classification, respectively 56.5, and 58.5,60.5,62.5,65.5,68.5,
Within 73.5,80,90,120 minutes, collect fraction, collect altogether ten fractions.
Embodiment 2
Utilize MARS-14 antibody column and the size exclusion chromatography technology HeLa cell to SILAC labelling
Culture fluid realizes enrichment and the qualification of secretory protein.
First collecting the culture fluid of the HeLa cell of SILAC labelling (K6), 500g is centrifugal to remove
The cell of residual, takes supernatant and carries out the centrifugal cell debris remained with removing of 3000g again.Then, take
The supernatant filter membrane of 0.45 μm carries out filtering to remove further cell debris, and collection flows through component.
Then, the process of MARS-14 antibody column is utilized to flow through component to remove high-abundance proteins therein more sharp
With size exclusion chromatography post (5 μm,) albumen is carried out the classification of molecular weight, and collect each
Fraction.Then, the albumen in each fraction adds the dithiothreitol, DTT 56 degrees Celsius of 100 μ L 0.1M
Degeneration reductase 12 hour, then lucifuge to add the iodoacetamide room temperature alkylated reaction half of 100 μ L 0.2M little
Time, it is subsequently added into the ammonium hydrogen carbonate of 1mL 50mM, adds trypsin 1:50, enzyme: albumen,
Mass ratio) 37 degrees Celsius hatch 16 hours, then with desalination post to sample desalination, finally carry out
Nano-ESI-LC-MS/MS mass spectral analysis.
MARS-14 antibody column processes culture fluid: first 20 μ L flow through component and 40 μ LBuffer A
(Agilent, 5185-5987) mixes, by liquid chromatographic system, with the flow velocity of 0.125mL/min
Flow through antibody column, and collection flows through liquid.Flow through liquid and be the culture fluid after eliminating high-abundance proteins.
Size exclusion chromatography carries out molecular-weight gradation to albumen: 100 μ L are flowed through component 1 × PBS
It is diluted 5 times, by liquid chromatographic system, flows through size exclusion color with the flow velocity of 0.3mL/min
Spectrum post, and collect 15, the fraction of 17,19,21,23,25,27,29,31,33 minutes, collect altogether ten fractions.
Embodiment 3
Utilize ProteoPrep20 antibody column and the size exclusion chromatography technology HeLa to SILAC labelling
Cell culture fluid realizes enrichment and the qualification of secretory protein.
First collecting the culture fluid of the HeLa cell of SILAC labelling (R10), 500g is centrifugal to remove
Remove the cell of residual, take supernatant and carry out the centrifugal cell debris remained with removing of 3000g again.Then,
The filter membrane taking supernatant 0.22 μm carries out filtering to remove further cell debris, and collection flows through group
Point.Then, utilize ProteoPrep20 antibody column to process and flow through component to remove high abundance egg therein
In vain, recycling size exclusion chromatography post (5 μm,) albumen is carried out the classification of molecular weight, and
Collect each fraction.Then, the albumen in each fraction adds the dithiothreitol, DTT of 100 μ L 0.1M
56 degrees Celsius of degeneration reductase 12s hour, then lucifuge adds the iodoacetamide room temperature alkyl of 100 μ L 0.2M
Change and react half an hour, be subsequently added into the ammonium hydrogen carbonate of 1mL 50mM, add trypsin 1:50,
Enzyme: albumen, mass ratio) 37 degrees Celsius hatch 16 hours, then with desalination post to sample desalination,
After carry out nano-ESI-LC-MS/MS mass spectral analysis.
ProteoPrep20 antibody column processes culture fluid: first 20 μ L flow through component and 40 μ L Buffer
A (Agilent, 5185-5987) mixes, by liquid chromatographic system, with the flow velocity of 0.125mL/min
Flow through antibody column, and collection flows through liquid.Flow through liquid and be the culture fluid after eliminating high-abundance proteins.
Size exclusion chromatography carries out molecular-weight gradation to albumen: 100 μ L are flowed through component 1 × PBS
It is diluted 5 times, by liquid chromatographic system, flows through size exclusion color with the flow velocity of 0.3mL/min
Spectrum post, and collect 15, the fraction of 17,19,21,23,25,27,29,31,33 minutes, collect altogether ten fractions.
Embodiment 4
Utilize SuperMix antibody column and Gelfree 8100 albumen classification instrument to SILAC labelling
HeLa cell culture fluid realizes enrichment and the qualification of secretory protein.
First collecting the culture fluid of the HeLa cell of SILAC labelling (R6), 500g is centrifugal to remove
The cell of residual, takes supernatant and carries out the centrifugal cell debris remained with removing of 3000g again.Then, take
The supernatant filter membrane of 0.45 μm carries out filtering to remove further cell debris, and collection flows through component.
Then, the process of SuperMix antibody column is utilized to flow through component to remove high-abundance proteins therein more sharp
With the Gelfree 8100 of Expedeon company, albumen is carried out the classification of molecular weight, and collects each evaporating
Point.Then, the albumen in each fraction adds the dithiothreitol, DTT 56 degrees Celsius change of 100 μ L 0.1M
Property reductase 12 hour, then lucifuge adds the iodoacetamide room temperature alkylated reaction half an hour of 100 μ L 0.2M,
It is subsequently added into the ammonium hydrogen carbonate of 1mL 50mM, adds trypsin 1:50, enzyme: albumen, matter
Amount than) 37 degrees Celsius hatch 16 hours, then with desalination post to sample desalination, finally carry out
Nano-ESI-LC-MS/MS mass spectral analysis.
SuperMix antibody column processes culture fluid: first 20 μ L flow through component and 40 μ L Buffer A
(Agilent, 5185-5987) mixes, by liquid chromatographic system, with the flow velocity of 0.125mL/min
Flow through antibody column, and collection flows through liquid.Flow through liquid and be the culture fluid after eliminating high-abundance proteins.
Gelfree 8100 albumen staged care condition: according to the operation instruction of instrument, takes 20 μ L
SampleBuffer and 10 μ L Running Buffer (producer provides by instrument), two sulfur of 8 μ L 1M
Threitol and the 112 pending sample mix of μ L are placed in the sample cell of instrument, the program recommended according to instrument
Voltage arranges and sample is carried out classification, respectively 56.5, and 58.5,60.5,62.5,65.5,68.5,
Within 73.5,80,90,120 minutes, collect fraction, collect altogether ten fractions.
Embodiment 5
Utilize ProteoMiner equalizer material and 3100Offgel Fractionator albumen classification instrument
HeLa cell culture fluid to SILAC labelling realizes enrichment and the qualification of secretory protein.
First collecting the culture fluid of the HeLa cell of SILAC labelling (K6), 500g is centrifugal to remove
The cell of residual, takes supernatant and carries out the centrifugal cell debris remained with removing of 3000g again.Then, take
The supernatant filter membrane of 0.22 μm carries out filtering to remove further cell debris, and collection flows through component.
Then, ProteoMiner equalizer processes is utilized to flow through component to remove high-abundance proteins therein simultaneously
Enrichment low-abundance protein, albumen isoelectric point, IP is entered by recycling 3100Offgel Fractionator classification instrument
The classification of row molecular weight, and collect each fraction.Then, the albumen in each fraction adds 100 μ L 0.1
56 degrees Celsius of degeneration reductase 12s hour of the dithiothreitol, DTT of M, then lucifuge adds the iodine of 100 μ L 0.2M
Acetamide room temperature alkylated reaction half an hour, it is subsequently added into the ammonium hydrogen carbonate of 1mL 50mM, adds
Trypsin 1:50, enzyme: albumen, mass ratio) 37 degrees Celsius hatch 16 hours, then use desalination
Post, to sample desalination, finally carries out nano-ESI-LC-MS/MS mass spectral analysis.
ProteoMiner equalizer processes condition: first the albumen of 200 μ L 60mg/ml is flowed through component
With 100mg ProteoMiner equalizer material incubated at room under the conditions of 10mM HEPES pH=7.0
2h.After having hatched, flush three times with 10mM HEPES, with 300 μ L eluents (4%SDS,
25mM DTT) under the conditions of 95 DEG C, hatch 15min, then carry out 500g and be centrifuged eluted product.
Eluent is the culture fluid after eliminating high-abundance proteins.
3100Offgel Fractionator albumen staged care condition: according to the operation instruction of instrument, take 2.88mL
1.25 × OFFGEL storing solution (producer provides by instrument) and 0.72mL water, mix with pending sample
Closing the sample cell being placed in instrument, use the adhesive tape of pH 3-10, the voltage recommended according to instrument arranges (200
V-1500V) sample is carried out classification in adhesive tape, collect altogether ten fractions.
Embodiment 6
Utilize ProteoPrep20 and 3100Offgel Fractionator albumen classification instrument that SILAC is marked
The HeLa cell culture fluid of note realizes enrichment and the qualification of secretory protein.
First collecting the culture fluid of the HeLa cell of SILAC labelling (K6, R10), 500g is centrifuged
To remove the cell of residual, take supernatant and carry out the centrifugal cell debris remained with removing of 3000g again.Connect
, the filter membrane taking supernatant 0.45 μm carries out filtering to remove further cell debris, and collection flows through
Component.Then, ProteoPrep20 process is utilized to flow through component same to remove high-abundance proteins therein
Time enrichment low-abundance protein, recycling 3100Offgel Fractionator classification instrument to albumen isoelectric point, IP
Carry out the classification of molecular weight, and collect each fraction.Then, the albumen in each fraction adds 100 μ L
56 degrees Celsius of degeneration reductase 12s hour of the dithiothreitol, DTT of 0.1M, then lucifuge adds 100 μ L 0.2M's
Iodoacetamide room temperature alkylated reaction half an hour, it is subsequently added into the ammonium hydrogen carbonate of 1mL 50mM, then adds
Enter trypsin 1:50, enzyme: albumen, mass ratio) 37 degrees Celsius hatch 16 hours, then with removing
Salt plug, to sample desalination, finally carries out nano-ESI-LC-MS/MS mass spectral analysis.
ProteoPrep20 antibody column processes culture fluid: first 20 μ L flow through component and 40 μ L Buffer
A (Agilent, 5185-5987) mixes, by liquid chromatographic system, with the flow velocity of 0.125mL/min
Flow through antibody column, and collection flows through liquid.Flow through liquid and be the culture fluid after eliminating high-abundance proteins.
3100 Offgel Fractionator albumen staged care conditions: according to the operation instruction of instrument, take
2.88mL 1.25 × OFFGEL storing solution (producer provides by instrument) and 0.72mL water, and wait to locate
Reason sample mix is placed in the sample cell of instrument, uses the adhesive tape of pH 3-10, the voltage recommended according to instrument
(200V-1500V) is set sample is carried out classification in adhesive tape, collect altogether ten fractions.
Claims (6)
1. one kind contains the sample pretreating method of secretory protein in serum free culture system liquid, it is characterised in that:
Take the cell culture fluid containing serum, by centrifugation, filtration treatment, then utilize high-abundance proteins successively
Removal technology and albumen classification technique process, and it is carried out enzymolysis.
The most in accordance with the method for claim 1, it is characterised in that: the described cell containing serum is cultivated
Liquid be containing 1%-30% hyclone and for cell cultivate SILAC culture fluid；Above-mentioned SILAC trains
Nutrient solution is containing isotope-labeled lysine, arginine, leucine, isoleucine, methionine,
The cell culture fluid of the combination of one or two or more kinds in agedoite.
The most in accordance with the method for claim 1, it is characterised in that: described centrifugal, filtration treatment,
Being specially the centrifugal force first with 300-700g and remove cell, centrifugation time is 1-60 minute；Take
The clear centrifugal force using 2000-5000g again removes cell debris, and centrifugation time is 1-60 minute, takes
Clear with the membrane filtration of 0.1-1.0 μm, take filtrate.
The most in accordance with the method for claim 1, it is characterised in that: described high-abundance proteins removes skill
Art is one or both of antibody column removal technology or protein balancing technique；Above-mentioned antibody column removes skill
Art is to utilize several immobilized antibody that its specific high-abundance proteins realizes specificity capture to reach
The purpose removed；Above-mentioned protein balancing technique is to utilize specific material that protein is realized equilibrium,
To reduce high-abundance proteins content, improve low-abundance protein content.
The most in accordance with the method for claim 1, it is characterised in that: described albumen classification technique is egg
One or both of white isoelectric point, IP classification technique or molecular weight of albumen classification technique；Above-mentioned albumen isoelectric point, IP
Classification technique is that it is separated by the isoelectric point, IP character utilizing protein different；Above-mentioned molecular weight of albumen
Classification technique is that it is separated by the character utilizing protein different molecular weight.
The most in accordance with the method for claim 1, it is characterised in that: described enzymolysis is that albumen is at pancreas egg
Under a kind of or effect of any two kinds of white enzyme, serine protease or protein incision enzyme, by albumen water
Solve as peptide fragment；The addition of enzyme is by enzyme: 1:1-1:200 based on albumen quality ratio；Enzymatic hydrolysis condition is 20-50
Degree Celsius lower enzymolysis 1-48 hour.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510387358.9A CN106324249A (en) | 2015-07-03 | 2015-07-03 | Sample pretreatment method of secretory proteins in culture medium with serum |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510387358.9A CN106324249A (en) | 2015-07-03 | 2015-07-03 | Sample pretreatment method of secretory proteins in culture medium with serum |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN106324249A true CN106324249A (en) | 2017-01-11 |
Family
ID=57726469
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510387358.9A Pending CN106324249A (en) | 2015-07-03 | 2015-07-03 | Sample pretreatment method of secretory proteins in culture medium with serum |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106324249A (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2309262A1 (en) * | 2009-10-06 | 2011-04-13 | Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. | Method for quantifying biomolecules |
| CN104170052A (en) * | 2012-04-02 | 2014-11-26 | 塞莫费雪科学（不来梅）有限公司 | Method and apparatus for improved quantitation by mass spectrometry |
-
2015
- 2015-07-03 CN CN201510387358.9A patent/CN106324249A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2309262A1 (en) * | 2009-10-06 | 2011-04-13 | Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. | Method for quantifying biomolecules |
| CN104170052A (en) * | 2012-04-02 | 2014-11-26 | 塞莫费雪科学（不来梅）有限公司 | Method and apparatus for improved quantitation by mass spectrometry |
Non-Patent Citations (3)
| Title |
|---|
| M.COLZANI ET AL.: "Metabolic Labeling and Protein Linearization Technology Allow the Study of Proteins Secreted by Cultured Cells in Serum-Containing Media", 《JOURNAL OF PROTEOME RESEARCH》 * |
| 李贤煜等: "等电聚焦预分离用于肝癌细胞分泌蛋白质组的分级与鉴定", 《色谱》 * |
| 黄凌瑾等: "非小细胞肺癌细胞分泌蛋白谱的鉴定", 《肿瘤研究与临床》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Silveira et al. | Separation of phycocyanin from Spirulina platensis using ion exchange chromatography | |
| KR20150125714A (en) | Continuous purification of therapeutic proteins | |
| Gomes et al. | Synergy of human platelet-derived extracellular vesicles with secretome proteins promotes regenerative functions | |
| Sharma et al. | Single‐step purification and characterization of antifreeze proteins from leaf and berry of a freeze‐tolerant shrub seabuckthorn (Hippophae rhamnoides) | |
| Xu et al. | Advances about the roles of membranes in cotton fiber development | |
| Luttmann et al. | Sequential/parallel production of potential Malaria vaccines–A direct way from single batch to quasi-continuous integrated production | |
| Hogwood et al. | An ultra scale‐down approach identifies host cell protein differences across a panel of mAb producing CHO cell line variants | |
| CN104152436B (en) | DNA isolation and purification methods and its kit | |
| Parsons et al. | Separation of the plant Golgi apparatus and endoplasmic reticulum by free-flow electrophoresis | |
| Tan et al. | Data-independent acquisition-based proteome and phosphoproteome profiling reveals early protein phosphorylation and dephosphorylation events in arabidopsis seedlings upon cold exposure | |
| Morales-Sanfrutos et al. | Unraveling the complexity of the extracellular vesicle landscape with advanced proteomics | |
| Knecht et al. | An introduction to analytical challenges, approaches, and applications in mass spectrometry–based secretomics | |
| Aguado et al. | Isolation of lysosomes from mammalian tissues and cultured cells | |
| Fröhlich et al. | Deep insights into the plant proteome by pretreatment with combinatorial hexapeptide ligand libraries | |
| CN106324249A (en) | Sample pretreatment method of secretory proteins in culture medium with serum | |
| Kim et al. | Combined analysis of the time-resolved transcriptome and proteome of plant pathogen Xanthomonas oryzae pv. oryzae | |
| Girondé et al. | A comparative study of proteolytic mechanisms during leaf senescence of four genotypes of winter oilseed rape highlighted relevant physiological and molecular traits for NRE improvement | |
| CN109053877B (en) | Method for extracting apolipoprotein B from serum and apolipoprotein B | |
| Hamzelou et al. | Utilizing proteomics to identify and optimize microalgae strains for high-quality dietary protein: A review | |
| Chen et al. | A simple method of catalase purification for the undergraduate experimental course | |
| Esteve et al. | Analytical approaches for the characterization and identification of olive (Olea europaea) oil proteins | |
| CN101698680A (en) | PEG-gelchromatography combined purification method for egg yolk immunoglobulins (IgY) | |
| CN112574296A (en) | Separation and purification method of IVIg-simulated multi-human-part mixed human plasma IgG sample | |
| CN113666988A (en) | Preparation method and application of N-terminal structural domain of novel coronavirus nucleocapsid protein | |
| Yamaguchi et al. | Localization of vanabins, vanadium-binding proteins, in the blood cells of the vanadium-rich ascidian, Ascidia sydneiensis samea |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20170111 |