CA3123719A1 - Method to perform limited two dimensional separation of proteins and other biologicals - Google Patents
Method to perform limited two dimensional separation of proteins and other biologicals Download PDFInfo
- Publication number
- CA3123719A1 CA3123719A1 CA3123719A CA3123719A CA3123719A1 CA 3123719 A1 CA3123719 A1 CA 3123719A1 CA 3123719 A CA3123719 A CA 3123719A CA 3123719 A CA3123719 A CA 3123719A CA 3123719 A1 CA3123719 A1 CA 3123719A1
- Authority
- CA
- Canada
- Prior art keywords
- separation
- separation channel
- channel
- zones
- proteins
- 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
- 238000000926 separation method Methods 0.000 title claims abstract description 108
- 238000000034 method Methods 0.000 title claims abstract description 28
- 102000004169 proteins and genes Human genes 0.000 title claims description 40
- 108090000623 proteins and genes Proteins 0.000 title claims description 40
- 229960000074 biopharmaceutical Drugs 0.000 title description 3
- 239000000126 substance Substances 0.000 claims abstract description 25
- 238000001514 detection method Methods 0.000 claims abstract description 12
- 239000000203 mixture Substances 0.000 claims description 15
- 238000001155 isoelectric focusing Methods 0.000 claims description 14
- 239000012530 fluid Substances 0.000 claims description 9
- 239000003792 electrolyte Substances 0.000 claims description 8
- 238000007873 sieving Methods 0.000 claims description 8
- 230000005684 electric field Effects 0.000 claims description 7
- 238000003384 imaging method Methods 0.000 claims description 7
- 239000000959 ampholyte mixture Substances 0.000 claims description 6
- 239000012491 analyte Substances 0.000 claims description 6
- 229920000609 methyl cellulose Polymers 0.000 claims description 6
- 239000001923 methylcellulose Substances 0.000 claims description 6
- 235000010981 methylcellulose Nutrition 0.000 claims description 6
- 230000004888 barrier function Effects 0.000 claims description 5
- 239000000872 buffer Substances 0.000 claims description 4
- 102000014914 Carrier Proteins Human genes 0.000 claims description 3
- 108010078791 Carrier Proteins Proteins 0.000 claims description 3
- 230000004044 response Effects 0.000 claims description 3
- 238000012544 monitoring process Methods 0.000 claims 5
- 239000000523 sample Substances 0.000 claims 5
- 239000012488 sample solution Substances 0.000 claims 4
- 230000001939 inductive effect Effects 0.000 claims 2
- 230000003287 optical effect Effects 0.000 claims 2
- 230000002706 hydrostatic effect Effects 0.000 claims 1
- 238000001962 electrophoresis Methods 0.000 abstract description 7
- 238000002347 injection Methods 0.000 abstract description 5
- 239000007924 injection Substances 0.000 abstract description 5
- 238000000533 capillary isoelectric focusing Methods 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 14
- 239000003550 marker Substances 0.000 description 12
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 9
- 102000018690 Trypsinogen Human genes 0.000 description 9
- 108010027252 Trypsinogen Proteins 0.000 description 9
- 238000001502 gel electrophoresis Methods 0.000 description 8
- 102000004338 Transferrin Human genes 0.000 description 6
- 108090000901 Transferrin Proteins 0.000 description 6
- LNAZSHAWQACDHT-XIYTZBAFSA-N (2r,3r,4s,5r,6s)-4,5-dimethoxy-2-(methoxymethyl)-3-[(2s,3r,4s,5r,6r)-3,4,5-trimethoxy-6-(methoxymethyl)oxan-2-yl]oxy-6-[(2r,3r,4s,5r,6r)-4,5,6-trimethoxy-2-(methoxymethyl)oxan-3-yl]oxyoxane Chemical compound CO[C@@H]1[C@@H](OC)[C@H](OC)[C@@H](COC)O[C@H]1O[C@H]1[C@H](OC)[C@@H](OC)[C@H](O[C@H]2[C@@H]([C@@H](OC)[C@H](OC)O[C@@H]2COC)OC)O[C@@H]1COC LNAZSHAWQACDHT-XIYTZBAFSA-N 0.000 description 5
- 102000036675 Myoglobin Human genes 0.000 description 5
- 108010062374 Myoglobin Proteins 0.000 description 5
- 229960002900 methylcellulose Drugs 0.000 description 5
- 230000008569 process Effects 0.000 description 5
- 239000012581 transferrin Substances 0.000 description 4
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 3
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- 230000002378 acidificating effect Effects 0.000 description 3
- 238000005370 electroosmosis Methods 0.000 description 3
- 150000002500 ions Chemical class 0.000 description 3
- 230000007246 mechanism Effects 0.000 description 3
- 235000011007 phosphoric acid Nutrition 0.000 description 3
- 238000005515 capillary zone electrophoresis Methods 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000012512 characterization method Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000007791 liquid phase Substances 0.000 description 2
- 230000005012 migration Effects 0.000 description 2
- 238000013508 migration Methods 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- 108010042653 IgA receptor Proteins 0.000 description 1
- 102100034014 Prolyl 3-hydroxylase 3 Human genes 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000001483 mobilizing effect Effects 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 230000002028 premature Effects 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 230000004845 protein aggregation Effects 0.000 description 1
- 230000006920 protein precipitation Effects 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000000539 two dimensional gel electrophoresis Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/416—Systems
- G01N27/447—Systems using electrophoresis
- G01N27/44704—Details; Accessories
- G01N27/44717—Arrangements for investigating the separated zones, e.g. localising zones
- G01N27/44721—Arrangements for investigating the separated zones, e.g. localising zones by optical means
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/416—Systems
- G01N27/447—Systems using electrophoresis
- G01N27/44756—Apparatus specially adapted therefor
- G01N27/44795—Isoelectric focusing
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/74—Optical detectors
- G01N2030/746—Optical detectors detecting along the line of flow, e.g. axial
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
- G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
- G01N2030/8813—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials
- G01N2030/8831—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials involving peptides or proteins
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Electrochemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Peptides Or Proteins (AREA)
Abstract
A method and apparatus are provided for performing capillary isoelectric focusing followed by mobilization of the focused zones by induced hydrodynamic flow or chemical mobilization. These two dimensions of separation are integrated with real-time whole-channel electrophoresis detection and automatic sample injection to achieve a separation resolution superior to that obtainable using known orthogonal capillary two dimensional arrangements.
Description
METHOD TO PERFORM LIMITED TWO DIMENSIONAL SEPARATION
OF PROTEINS AND OTHER BIOLOGICALS
BACKGROUND OF THE INVENTION
[0001] The present invention is in the technical field of two-dimensional separation of proteins and other biologicals and relates particularly to apparatus and a method * for the rapid and reproducible separation of species in a liquid medium.
OF PROTEINS AND OTHER BIOLOGICALS
BACKGROUND OF THE INVENTION
[0001] The present invention is in the technical field of two-dimensional separation of proteins and other biologicals and relates particularly to apparatus and a method * for the rapid and reproducible separation of species in a liquid medium.
[0002] The separation and characterization of proteins is ubiquitous throughout the life sciences. Two of the most popular electrophoresis separation techniques are: 1) gel isoelectric focusing (IEF), where the separation mechanism is based on protein surface charge providing isoelectric point (pI) separation and 2) sodium dodecyl sulfate (SDS) gel electrophoresis where the separation mechanism is based on molecular weight (MW). These two techniques are most commonly performed individually.
[0003] Isoelectric focusing (IEF) is a special electrophoretic technique for separating amphoteric substances such as peptides and proteins in an electric field, across which there is both voltage and a pH gradient, acidic in the region of the anode and alkaline near the cathode. Each substance in the mixture will migrate to a position in the separation column where the surrounding pH corresponds to its isoelectric point.
There, in zwitterion form with no net charge, molecules of that substance cease to move in the electric field. Different amphorteric substances are thereby focused into narrow stationary bands.
There, in zwitterion form with no net charge, molecules of that substance cease to move in the electric field. Different amphorteric substances are thereby focused into narrow stationary bands.
[0004] In IEF separation, it is well known that proteins having molecular weight differences or conformational differences may possess similar pI values and therefore focus at the same location. In order to then separate these co-focused proteins, a technique called two-dimensional (2D) gel electrophoresis has been employed.
Date Recue/Date Received 2021-06-30 gel electrophoresis combines two orthogonal separation techniques - gel IEF
and SDS
gel - to create a technique that dramatically increases separation resolution and provides for the separation of co-focused IEF protein zones. 2D gel electrophoresis is generally carried out in a polyacrylamide slab gel and although it has become a workhorse in the field of proteomics, owing to the high degree of resolution which can be obtained thereby, it is very labour-intensive, time consuming and non-quantitative.
Moreover, although 2D gel electrophoresis does afford the highest degree of molecular weight resolution of known electrophoretic separation techniques, it has not yet been possible to automate that process nor quantify the resolved component proteins or other analytes. These and other drawbacks have motivated researchers to combine two orthogonal separation techniques in the liquid phase, using a capillary or coplanar microchannel format. While these are necessarily "limited resolution"
techniques, relative to 2D gel electrophoresis, they are much simpler and faster to use and are of adequate resolution for many purposes.
Date Recue/Date Received 2021-06-30 gel electrophoresis combines two orthogonal separation techniques - gel IEF
and SDS
gel - to create a technique that dramatically increases separation resolution and provides for the separation of co-focused IEF protein zones. 2D gel electrophoresis is generally carried out in a polyacrylamide slab gel and although it has become a workhorse in the field of proteomics, owing to the high degree of resolution which can be obtained thereby, it is very labour-intensive, time consuming and non-quantitative.
Moreover, although 2D gel electrophoresis does afford the highest degree of molecular weight resolution of known electrophoretic separation techniques, it has not yet been possible to automate that process nor quantify the resolved component proteins or other analytes. These and other drawbacks have motivated researchers to combine two orthogonal separation techniques in the liquid phase, using a capillary or coplanar microchannel format. While these are necessarily "limited resolution"
techniques, relative to 2D gel electrophoresis, they are much simpler and faster to use and are of adequate resolution for many purposes.
[0005] It is known to combine capillary or channel isoelectric focusing (cIEF) with non-porous reverse phase microliquid chromatography (RPLC) in a two-dimensional layout, to obtain useful online detection and quantitation. However, the interface between the first and second separation dimension has hitherto been carried out only at the outlet end of the IEF separation capillary or channel. It is known that the separation and pH gradient obtained in cIEF may be disturbed when mobilizing focused protein zones to reach the outlet end. A as result, it is more challenging to transfer separated zones from the first separation dimension to the second separation dimension in the orthogonal capillary or microchannel format than in apparatus for 2D
gel electrophoresis. Fluid connections and for control of nanoliter volumes are required, making for complex analytical design and operation.
BRIEF SUMMARY OF THE INVENTION
Date Recue/Date Received 2021-06-30
gel electrophoresis. Fluid connections and for control of nanoliter volumes are required, making for complex analytical design and operation.
BRIEF SUMMARY OF THE INVENTION
Date Recue/Date Received 2021-06-30
[0006] This invention describes improved method and apparatus for carrying out limited electrophoretic separation in the liquid phase. The objective of the invention is to provide a simple method and apparatus for limited "2D" separation using both capillary or channel IEF separation and capillary zone electrophoresis (CZE) separation within the same capillary or channel. The present invention also integrates real-time, whole-channel electrophoresis detection with automatic sample injection, automatic cIEF separation, separation zone manipulation and on-line electrolyte selection, to achieve a separation resolution superior to that obtained using an orthogonal capillary arrangement.
[0007] The quotation marks about "2D" above reflect the fact that the present invention uses two different and sequential electrophoretic techniques, but not orthogonal capillaries as in the known arrangements described above. The term "2D"
is, a convenient shorthand term for designating a method and apparatus employing two-stage electrophoretic separation, and will be used in the remainder of the specification and in the claims without quotation marks.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] Figure 1 is a schematic representation of a first embodiment of apparatus for performing limited 2D separation using electrophoresis and controlled hydrodynamic flow.
[0011] Figure 2 illustrates schematically a physiochemical mechanism postulated to explain the separation of proteins in the presence of a hydrodynamic flow as in the method of the invention.
Date Recue/Date Received 2021-06-30 [0012] Figure 3 is a schematic representation of a second embodiment of apparatus according to the invention for performing limited 2D separation using electrophoresis and chemical mobilization.
[0013] Figure 4 illustrates graphically the separation of two proteins having the same pI value but different charge responses to pH, using the method of the invention.
[0014] Figure 5 illustrates graphically the results of a separation effected by using apparatus according to the first embodiment of the invention, showing a single peak of tryptosinogen and pI Marker 9.46 mixture when hydrodynamic flow is minimized, and split peak of tryptosinogen and pI Marker 9.46 when hydrodynamic flow is toward the cathode.
[0015] Figure 6 illustrates graphically the results of a separation effected by apparatus according to the second embodiment of the invention, showing two peaks of transferrin prior to anodic mobilization and four peaks of transferring subsequent to anodic chemical mobilization.
DETAILED DESCRIPTION OF THE INVENTION
[0016] Figure 1 shows a first embodiment of the apparatus. A microfluid device is provided, including an anolyte tank 10 and a catholyte tank 12 such that electrolyes in the tanks are isolated from the sample mixture by ion conductive barriers 14 (such as semipereamble membranes). A high voltage supply connected across two electrodes that are immersed in the respective tanks. A CCD imaging camera 20 is focused so that it can detect light passing through or emitted from the entire length of a horizontal capillary separation channel 22. The camera 20 is able to display and capture pictures in real-time, or at least very rapidly. A
light source and collimation means (not shown) are provided for applying a sheet of light (arrows Date Recue/Date Received 2021-06-30 L) to pass through or emit from the entire length of separation channel 22. A
real time CCD sensor camera/sensor arrangement like that used with the apparatus of the present invention is described in more detail in US patent No. 6,852,206, having a common inventor and the same assignee as the present application. US patent 6,852,206 discloses detection and measurement apparatus of analyte separation zones in a capillary.
(0017] A switch valve 24 is connected to the microfluidic device such that an inlet flow channel portion 26 at one end of the separation channel may be selectively connected to either an autosampler 28 for sample injection, or to the fluid medium contents of an inlet vial 30. A hydrodynamic flow across separation channel 22 can be induced and controlled by vertical up or down fine-control motion of a hydrodynamic flow vial 32 containing fluid medium, the contents of which are connected by means of hydrodynamic flow control valve 33 with an outlet flow channel portion 34 of the separation channel, [0018] With the switch valve 24 position set for fluid connection of the inlet channel portion 26 of the separation channel to the autosampler 28, and with a shut-off valvefor autosampler connection tube 29 open, a sample containing a mixture of proteins , carrier ampholytes and a sieving solution such as methyl cellulose is injected into the separation channel by the autosampler until the sample mixture volume fills the separation channel to overflow. The position of the switch valve is then set to connect the inlet vial with the separation channel and the high voltage is turned on by means of HV switch 36. An electric field is thereby established across the separation channel and a linear pH gradient is formed by the carrier ampholytes.
The cIEF process begins and upon completion, proteins are focused and separated into zones according to their pI when both electro-osmotic flow and hydrodynamic flow are stable. The entire IEF process is continuously monitored and the images of the separation trace are continuously captured (recorded) in real-time by the whole-channel CCD imaging camera of the CCD sensor unit. At this point, the first Date Recue/Date Received 2021-06-30 dimensional separation (cIEF) is complete and the second dimensional separation is initiated.
[0019] The second dimensional separation is applied to the IEF focused zones (proteins) by the application of a controlled hydrodynamic flow. The hydrodynamic flow is induced by a microgravitational force arising in the separation channel 22 resulting from the finely controlled up or down motion of the hydrodynamic flow vial.
When hydrodynamic flow is introduced into the separation channel following IEF
focusing, the pH gradient will be affected and additional sample mixture will enter the separation channel. As more sample mixture is continuously injected into the separation channel owing to the hydrodynamic flow, the focused zones at the far end of the separation channel (along the direction of hydrodynamic flow) are continuously pushed out. For example, if the outlet vial 32 is raised slightly, then the hydrodynamic flow direction proceeds from the anodic (outlet end) to the cathodic end (inlet end). More sample mixture is introduced from the anodic end, and the most basic zones focused at the cathodic end will be pushed out of the separation capillary (over the ion conductive barrier area, see Figure 2). Since this hydrodynamic flow coexists with an electric field, the separation zone resolution and shape is preserved when the hydrodynamic flow is limited and carefully controlled and the newly injected sample mixture ampholytes are focused into their pI position.
The movement of relatively larger molecular weight proteins (protein A in Fig 2) is slower than that of smaller ones (protein B in Fig 2) in a sieving solution such as methylcellulose. As a result, a limited second dimensional separation of cIEF
zones (proteins) due to mass difference is achieved. Again, the entire second dimension separation process is continuously monitored and the images of the separation trace are continuously captured (recorded) in real-time by the whole-channel, CCD
imaging camera.
[0020] Figure 3 shows a second embodiment of the apparatus. The same reference numerals are used to indicate components corresponding to those of the Date Recue/Date Received 2021-06-30 first apparatus embodiment (Fig. 1). The microfluid device contains an analyte tank 10, a catholyte tank 12 and a chemical mobilization tank 38. The electrolyes in the three tanks are isolated from the sample mixture by ion conductive barriers 14. High voltage supply is connected at one end to an electrode immersed in the anolyte tank and at the other end to HV switch 36 such that connection can be made to either an electrode immersed in the catholyte tank or an electrode immersed in the chemical mobilization tank. Real time CCD sensor 20 is focused such that it can detect light (arrows L) passing through or emitted from the entire length of separation channel 22 and the camera is able to display and capture pictures in real-time, or at least very rapidly. Means (not shown) are provided in both the first and second embodiments of the invention for projecting a sheet of light to pass through or emit from the entire length of the separation channel. As with the first embodiment described above switch valve 24 is connected to the microfluidic device such that the inlet flow channel 26 may be connected to either autosampler 18 for sample injection or to an inlet vial 30. The end of the outlet channel is immersed in an outlet vial.
[0021] The anolyte, catholyte and chemical mobilization tanks (10, 12,38) are filled with appropriate electrolytes and, with the switch valve position set for connection between the inlet of the separation channel and the autosampler and the shut-off valvle to capillary section 29 open, a sample containing a mixture of proteins , carrier ampholytes and a sieving solution such as methyl cellulose solution is injected into the separation channel by the autosampler until the sample mixture volume fills the separation channel to overflow. The switch valve position is then set for connection between inlet vial 30 and separation channel 22, the high voltage is turned on and the switch valve 24 is set such that the catholyte electrode is contacted, an electric field established across the separation channel, and a linear pH
gradient is formed by the carrier ampholytes. The cIEF process begins and upon completion, proteins are focused and separated into zones according to their pI when both electro-osmotic flow and hydrodynamic flow are well controlled. The entire cIEF
process is continuously monitored and the images of the separation trace are Date Recue/Date Received 2021-06-30 continuously captured (recorded) in real-time by the whole-channel, CCD
imaging camera. At this point, the first dimensional separation (cIEF) is complete and the second dimensional separation begins.
[0022] The second dimensional separation is achieved in this second embodiment of the apparatus, not by controlled hydrodynamic pressure but by chemical mobilization of the cIEF focused zones. An electric switch that is selectively operable to connect to anolyte electrode or the catholyte electrode is changed to connect to the chemical mobilization solution upon completion of cIEF.
Mobilization of the focused zones will then occur. It is known that when non-acid solution is used as the anolyte, focused cIEF zones will migrate towards the anode (anodic mobilization).
Whereas when non-base solution is used as the catholyte, focused cIEF zones will migrate towards the cathode (cathodic mobilization). Therefore, anodic mobilization may be achieved by switching the high voltage contact to the anode from the acid solution tank to the chemical mobilization tank that contains non-acid solution, or cathodic mobilization may be achieved by switching the high voltage contact to the cathode from the base solution tank to the chemical mobilization tank that contains non-base solution.
[0023] The rate of migration due to chemical mobilization is determined by the charge-to-mass ratio of the protein and the mobility of the protein in a specific sieving solution. For example, two exemplary proteins with the same pI value have different rates of migration in response to a pH change (Figure 4). As a result, these two proteins will not experience the same rate of motion during chemical mobilization. In addition, when this movement is carried out in a sieving solution, proteins with different molecular weight or shape (conformation) may have different mobility. Therefore, proteins with the same pI, but have different mobility change with pH or different molecular weights or conformation can be separated with limited 2D separation of cIEF zones using chemical mobilization. Again, the entire second dimension separation process is continuously monitored and the images of the
is, a convenient shorthand term for designating a method and apparatus employing two-stage electrophoretic separation, and will be used in the remainder of the specification and in the claims without quotation marks.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] Figure 1 is a schematic representation of a first embodiment of apparatus for performing limited 2D separation using electrophoresis and controlled hydrodynamic flow.
[0011] Figure 2 illustrates schematically a physiochemical mechanism postulated to explain the separation of proteins in the presence of a hydrodynamic flow as in the method of the invention.
Date Recue/Date Received 2021-06-30 [0012] Figure 3 is a schematic representation of a second embodiment of apparatus according to the invention for performing limited 2D separation using electrophoresis and chemical mobilization.
[0013] Figure 4 illustrates graphically the separation of two proteins having the same pI value but different charge responses to pH, using the method of the invention.
[0014] Figure 5 illustrates graphically the results of a separation effected by using apparatus according to the first embodiment of the invention, showing a single peak of tryptosinogen and pI Marker 9.46 mixture when hydrodynamic flow is minimized, and split peak of tryptosinogen and pI Marker 9.46 when hydrodynamic flow is toward the cathode.
[0015] Figure 6 illustrates graphically the results of a separation effected by apparatus according to the second embodiment of the invention, showing two peaks of transferrin prior to anodic mobilization and four peaks of transferring subsequent to anodic chemical mobilization.
DETAILED DESCRIPTION OF THE INVENTION
[0016] Figure 1 shows a first embodiment of the apparatus. A microfluid device is provided, including an anolyte tank 10 and a catholyte tank 12 such that electrolyes in the tanks are isolated from the sample mixture by ion conductive barriers 14 (such as semipereamble membranes). A high voltage supply connected across two electrodes that are immersed in the respective tanks. A CCD imaging camera 20 is focused so that it can detect light passing through or emitted from the entire length of a horizontal capillary separation channel 22. The camera 20 is able to display and capture pictures in real-time, or at least very rapidly. A
light source and collimation means (not shown) are provided for applying a sheet of light (arrows Date Recue/Date Received 2021-06-30 L) to pass through or emit from the entire length of separation channel 22. A
real time CCD sensor camera/sensor arrangement like that used with the apparatus of the present invention is described in more detail in US patent No. 6,852,206, having a common inventor and the same assignee as the present application. US patent 6,852,206 discloses detection and measurement apparatus of analyte separation zones in a capillary.
(0017] A switch valve 24 is connected to the microfluidic device such that an inlet flow channel portion 26 at one end of the separation channel may be selectively connected to either an autosampler 28 for sample injection, or to the fluid medium contents of an inlet vial 30. A hydrodynamic flow across separation channel 22 can be induced and controlled by vertical up or down fine-control motion of a hydrodynamic flow vial 32 containing fluid medium, the contents of which are connected by means of hydrodynamic flow control valve 33 with an outlet flow channel portion 34 of the separation channel, [0018] With the switch valve 24 position set for fluid connection of the inlet channel portion 26 of the separation channel to the autosampler 28, and with a shut-off valvefor autosampler connection tube 29 open, a sample containing a mixture of proteins , carrier ampholytes and a sieving solution such as methyl cellulose is injected into the separation channel by the autosampler until the sample mixture volume fills the separation channel to overflow. The position of the switch valve is then set to connect the inlet vial with the separation channel and the high voltage is turned on by means of HV switch 36. An electric field is thereby established across the separation channel and a linear pH gradient is formed by the carrier ampholytes.
The cIEF process begins and upon completion, proteins are focused and separated into zones according to their pI when both electro-osmotic flow and hydrodynamic flow are stable. The entire IEF process is continuously monitored and the images of the separation trace are continuously captured (recorded) in real-time by the whole-channel CCD imaging camera of the CCD sensor unit. At this point, the first Date Recue/Date Received 2021-06-30 dimensional separation (cIEF) is complete and the second dimensional separation is initiated.
[0019] The second dimensional separation is applied to the IEF focused zones (proteins) by the application of a controlled hydrodynamic flow. The hydrodynamic flow is induced by a microgravitational force arising in the separation channel 22 resulting from the finely controlled up or down motion of the hydrodynamic flow vial.
When hydrodynamic flow is introduced into the separation channel following IEF
focusing, the pH gradient will be affected and additional sample mixture will enter the separation channel. As more sample mixture is continuously injected into the separation channel owing to the hydrodynamic flow, the focused zones at the far end of the separation channel (along the direction of hydrodynamic flow) are continuously pushed out. For example, if the outlet vial 32 is raised slightly, then the hydrodynamic flow direction proceeds from the anodic (outlet end) to the cathodic end (inlet end). More sample mixture is introduced from the anodic end, and the most basic zones focused at the cathodic end will be pushed out of the separation capillary (over the ion conductive barrier area, see Figure 2). Since this hydrodynamic flow coexists with an electric field, the separation zone resolution and shape is preserved when the hydrodynamic flow is limited and carefully controlled and the newly injected sample mixture ampholytes are focused into their pI position.
The movement of relatively larger molecular weight proteins (protein A in Fig 2) is slower than that of smaller ones (protein B in Fig 2) in a sieving solution such as methylcellulose. As a result, a limited second dimensional separation of cIEF
zones (proteins) due to mass difference is achieved. Again, the entire second dimension separation process is continuously monitored and the images of the separation trace are continuously captured (recorded) in real-time by the whole-channel, CCD
imaging camera.
[0020] Figure 3 shows a second embodiment of the apparatus. The same reference numerals are used to indicate components corresponding to those of the Date Recue/Date Received 2021-06-30 first apparatus embodiment (Fig. 1). The microfluid device contains an analyte tank 10, a catholyte tank 12 and a chemical mobilization tank 38. The electrolyes in the three tanks are isolated from the sample mixture by ion conductive barriers 14. High voltage supply is connected at one end to an electrode immersed in the anolyte tank and at the other end to HV switch 36 such that connection can be made to either an electrode immersed in the catholyte tank or an electrode immersed in the chemical mobilization tank. Real time CCD sensor 20 is focused such that it can detect light (arrows L) passing through or emitted from the entire length of separation channel 22 and the camera is able to display and capture pictures in real-time, or at least very rapidly. Means (not shown) are provided in both the first and second embodiments of the invention for projecting a sheet of light to pass through or emit from the entire length of the separation channel. As with the first embodiment described above switch valve 24 is connected to the microfluidic device such that the inlet flow channel 26 may be connected to either autosampler 18 for sample injection or to an inlet vial 30. The end of the outlet channel is immersed in an outlet vial.
[0021] The anolyte, catholyte and chemical mobilization tanks (10, 12,38) are filled with appropriate electrolytes and, with the switch valve position set for connection between the inlet of the separation channel and the autosampler and the shut-off valvle to capillary section 29 open, a sample containing a mixture of proteins , carrier ampholytes and a sieving solution such as methyl cellulose solution is injected into the separation channel by the autosampler until the sample mixture volume fills the separation channel to overflow. The switch valve position is then set for connection between inlet vial 30 and separation channel 22, the high voltage is turned on and the switch valve 24 is set such that the catholyte electrode is contacted, an electric field established across the separation channel, and a linear pH
gradient is formed by the carrier ampholytes. The cIEF process begins and upon completion, proteins are focused and separated into zones according to their pI when both electro-osmotic flow and hydrodynamic flow are well controlled. The entire cIEF
process is continuously monitored and the images of the separation trace are Date Recue/Date Received 2021-06-30 continuously captured (recorded) in real-time by the whole-channel, CCD
imaging camera. At this point, the first dimensional separation (cIEF) is complete and the second dimensional separation begins.
[0022] The second dimensional separation is achieved in this second embodiment of the apparatus, not by controlled hydrodynamic pressure but by chemical mobilization of the cIEF focused zones. An electric switch that is selectively operable to connect to anolyte electrode or the catholyte electrode is changed to connect to the chemical mobilization solution upon completion of cIEF.
Mobilization of the focused zones will then occur. It is known that when non-acid solution is used as the anolyte, focused cIEF zones will migrate towards the anode (anodic mobilization).
Whereas when non-base solution is used as the catholyte, focused cIEF zones will migrate towards the cathode (cathodic mobilization). Therefore, anodic mobilization may be achieved by switching the high voltage contact to the anode from the acid solution tank to the chemical mobilization tank that contains non-acid solution, or cathodic mobilization may be achieved by switching the high voltage contact to the cathode from the base solution tank to the chemical mobilization tank that contains non-base solution.
[0023] The rate of migration due to chemical mobilization is determined by the charge-to-mass ratio of the protein and the mobility of the protein in a specific sieving solution. For example, two exemplary proteins with the same pI value have different rates of migration in response to a pH change (Figure 4). As a result, these two proteins will not experience the same rate of motion during chemical mobilization. In addition, when this movement is carried out in a sieving solution, proteins with different molecular weight or shape (conformation) may have different mobility. Therefore, proteins with the same pI, but have different mobility change with pH or different molecular weights or conformation can be separated with limited 2D separation of cIEF zones using chemical mobilization. Again, the entire second dimension separation process is continuously monitored and the images of the
- 8 -Date Recue/Date Received 2021-06-30 separation trace are continuously captured (recorded) in real-time by the whole-channel, CCD imaging camera.
[0024] cIEF is a steady state technique. Focusing and separation of proteins is achieved when transitional peaks or zones converge into stationary zones.
However, if single-point detection is used, it is difficult to know the exact time when all proteins are focused, since the speed of protein focusing is affected by sample conditions such as: content of salt and carrier ampholytes in the sample, experimental conditions such as separation channel dimensions, electric field strength and electrolyte concentration. As a result, two transitional peaks or zones for one protein may be detected when the protein is not yet focused. Further, an abnormal peak may be observed due to protein aggregation or precipitation resulting from prolonged protein focusing. With whole-column detection, as used with the present invention, however, the separation and focusing of an individual protein can be monitored in real time, avoiding the problems of 2D separation of transitional peaks (premature focusing) and separation of precipitated proteins (over focusing). The pI value of the protein is calibrated and the second dimension separation is applied. With real-time, whole column detection, the protein separation can be monitored, providing better protein fingerprinting by allowing straightforward assignment of protein zones based on pI
and relative molecular weight differences.
Example 1: Induced Hydrodynamic Flow as Second Dimension of Separation [0025] Figure 5 illustrates hydrodynamic flow induced limited 2D
separation of protein trypsinogen and a small molecular weight pI marker. In this experiment, trypsinogen and a small molecular pI marker were mixed with 8% pH 3-10 Pharmalyte and 0.35% methylcellulose. The sample mixture was injected into a mm 100 pm inner diameter FC coated capillary with a micro autosampler.
Focusing was conducted at a focusing voltage of 3000 V, with 80 mM H3PO4 as anolyte and mM NaOH as catholyte. Detection was conducted with a real-time, whole column UV
[0024] cIEF is a steady state technique. Focusing and separation of proteins is achieved when transitional peaks or zones converge into stationary zones.
However, if single-point detection is used, it is difficult to know the exact time when all proteins are focused, since the speed of protein focusing is affected by sample conditions such as: content of salt and carrier ampholytes in the sample, experimental conditions such as separation channel dimensions, electric field strength and electrolyte concentration. As a result, two transitional peaks or zones for one protein may be detected when the protein is not yet focused. Further, an abnormal peak may be observed due to protein aggregation or precipitation resulting from prolonged protein focusing. With whole-column detection, as used with the present invention, however, the separation and focusing of an individual protein can be monitored in real time, avoiding the problems of 2D separation of transitional peaks (premature focusing) and separation of precipitated proteins (over focusing). The pI value of the protein is calibrated and the second dimension separation is applied. With real-time, whole column detection, the protein separation can be monitored, providing better protein fingerprinting by allowing straightforward assignment of protein zones based on pI
and relative molecular weight differences.
Example 1: Induced Hydrodynamic Flow as Second Dimension of Separation [0025] Figure 5 illustrates hydrodynamic flow induced limited 2D
separation of protein trypsinogen and a small molecular weight pI marker. In this experiment, trypsinogen and a small molecular pI marker were mixed with 8% pH 3-10 Pharmalyte and 0.35% methylcellulose. The sample mixture was injected into a mm 100 pm inner diameter FC coated capillary with a micro autosampler.
Focusing was conducted at a focusing voltage of 3000 V, with 80 mM H3PO4 as anolyte and mM NaOH as catholyte. Detection was conducted with a real-time, whole column UV
- 9 -Date Recue/Date Received 2021-06-30 detector. The hydrodynamic flow is controlled by the water level difference in the hydrodynamic flow vial and the inlet vial.
[0026] It can be seen that when hydrodynamic flow was minimized (i.e. under first dimension cIEF separation conditions), there were two peaks in the electrophorogram (trace a). The more acidic peak to the left of the electrophorogram (egram) contains the minor component of trypsinogen (pk 1) and the more basic peak to the right of the egram contains the major component of trypsinogen (pk 2) and the pI marker (pk3). When a hydrodynamic flow was introduced in the direction of the cathodic end (trace b), the minor component of trypsinogen (pk 1) further partially separated into two subcomponents, and the pI marker (pk 3) was partially separated from peak the major component of trypsinogen (pk 2). The pI marker (pk 3) moved more quickly to a more basic position than the major trypsinogen component (pk 2) due to its smaller molecular weight in a sieving solution.
When a hydrodynamic flow was introduced in the direction of the anodic end (trace c), again because of the smaller MW of the pI marker (pk 3) compared to that of the major component of trypsinogen (pk 2), the pI marker shifted more quickly to a more acidic position than that of the major component of trypsinogen.
Example 2: Chemical Mobilization as Second Dimension of Separation [0027] Figure 6 illustrates chemical mobilization induced limited 2D
separation of transferrin, myoglobin and a small molecular weight pI marker (pI 4.22). In this experiment, transferrin and myoglobin and the pI marker were mixed with 8% pH
Pharmalyte and 0.35% methylcellu lose. The sample mixture was injected into a mm 100 pm inner diameter FC coated capillary with a micro autosampler.
Focusing was conducted at a focusing voltage of 3000 V, with 80 mM H3PO4 as anolyte and mM NaOH as catholyte. Detection was conducted with a real-time, whole column UV
detector. For anodic mobilization (trace b), the anolyte was replaced with 100 mM
NaOH upon completion of cIEF focusing. For cathodic mobilization (trace c), the catholyte was replaced with 80 mM H3PO4 upon completion of focusing.
[0026] It can be seen that when hydrodynamic flow was minimized (i.e. under first dimension cIEF separation conditions), there were two peaks in the electrophorogram (trace a). The more acidic peak to the left of the electrophorogram (egram) contains the minor component of trypsinogen (pk 1) and the more basic peak to the right of the egram contains the major component of trypsinogen (pk 2) and the pI marker (pk3). When a hydrodynamic flow was introduced in the direction of the cathodic end (trace b), the minor component of trypsinogen (pk 1) further partially separated into two subcomponents, and the pI marker (pk 3) was partially separated from peak the major component of trypsinogen (pk 2). The pI marker (pk 3) moved more quickly to a more basic position than the major trypsinogen component (pk 2) due to its smaller molecular weight in a sieving solution.
When a hydrodynamic flow was introduced in the direction of the anodic end (trace c), again because of the smaller MW of the pI marker (pk 3) compared to that of the major component of trypsinogen (pk 2), the pI marker shifted more quickly to a more acidic position than that of the major component of trypsinogen.
Example 2: Chemical Mobilization as Second Dimension of Separation [0027] Figure 6 illustrates chemical mobilization induced limited 2D
separation of transferrin, myoglobin and a small molecular weight pI marker (pI 4.22). In this experiment, transferrin and myoglobin and the pI marker were mixed with 8% pH
Pharmalyte and 0.35% methylcellu lose. The sample mixture was injected into a mm 100 pm inner diameter FC coated capillary with a micro autosampler.
Focusing was conducted at a focusing voltage of 3000 V, with 80 mM H3PO4 as anolyte and mM NaOH as catholyte. Detection was conducted with a real-time, whole column UV
detector. For anodic mobilization (trace b), the anolyte was replaced with 100 mM
NaOH upon completion of cIEF focusing. For cathodic mobilization (trace c), the catholyte was replaced with 80 mM H3PO4 upon completion of focusing.
-10 -Date Recue/Date Received 2021-06-30 In Trace a, it can be seen that when electroosmotic flow and hydrodynamic flow are well controlled (i.e. under first dimension cIEF separation conditions), the transferrin protein is partially resolved into two peaks and a minor myoglobin peak (pk 1) is noted. Under anodic mobilization (trace b), the transferrin protein is now partially resolved into 4 peaks and the minor myoglobin component is partially resolved into 2 peaks (pk 1). When cathodic chemical mobilization was introduced (trace c), the two peaks of transferrin (trace a) are separated into two larger peaks and one smaller peak.
[0028] Neither chemical mobilization conditions produced any split or partially separation of the pI marker peak (pI 4.22) and the major myoglobin peak.
CONCLUSION
[0029] From the description and examples herein it will be seen that applicants' provides a rapid, reproducible and quantative limited 2D electrophoresis separation.
Channel or capillary-based electrophoresis, unlike 2D gel electrophoresis permits automatic sample injection. No sample transfer or handling is involved and either hydrodynamic flow or chemical mobilization can be used, since both can be well controlled. Applicants' arrangement allows "two-dimensional" electrophoresis to be carried out within a single separation channel and in a single analysis run.
The use of real time, whole channel image detection affords very good reproducibility in both qualitative and quantative characterization.
[0030] While the foregoing written description of the invention enables one of ordinary skill to make and use what is considered presently-to be the best mode thereof, those of ordinary skill will understand and appreciate the existence of variations, combinations, and equivalents of the specific embodiment, method, and examples herein. The invention should therefore not be limited by the above
[0028] Neither chemical mobilization conditions produced any split or partially separation of the pI marker peak (pI 4.22) and the major myoglobin peak.
CONCLUSION
[0029] From the description and examples herein it will be seen that applicants' provides a rapid, reproducible and quantative limited 2D electrophoresis separation.
Channel or capillary-based electrophoresis, unlike 2D gel electrophoresis permits automatic sample injection. No sample transfer or handling is involved and either hydrodynamic flow or chemical mobilization can be used, since both can be well controlled. Applicants' arrangement allows "two-dimensional" electrophoresis to be carried out within a single separation channel and in a single analysis run.
The use of real time, whole channel image detection affords very good reproducibility in both qualitative and quantative characterization.
[0030] While the foregoing written description of the invention enables one of ordinary skill to make and use what is considered presently-to be the best mode thereof, those of ordinary skill will understand and appreciate the existence of variations, combinations, and equivalents of the specific embodiment, method, and examples herein. The invention should therefore not be limited by the above
- 11 -Date Recue/Date Received 2021-06-30 described embodiment, method, and examples, but by all embodiments and methods within the scope and spirit of the invention as claimed.
- 12 -Date Recue/Date Received 2021-06-30
Claims (9)
1. Apparatus for the electrophoretic separation of amphoteric biological analytes in a solution in an ampholytic carrier, comprising:
(a) a horizontal capillary having a central separation channel portion, an integral inlet portion at one end thereof and an integral outlet channel portion at the other end thereof;
(b) at each end of said separation channel portion, an electrolyte tank including an ion conductive barrier separating fluid inside the tank from fluid in the separation column;
(c) means for filling the capillary with a sample of analyte solution comprising autosampler means, a reservoir for sample solution and switch valve means for selectively connecting said autosampler and said inlet reservoir to said inlet channel;
(d) means for establishing isoelectric focusing of analytes within said separation channel into zones comprising an electrode in each of said electrolyte tanks and a switchable high voltage supply connected across the electrodes immersed in the electrolyte in said tanks;
(e) means for applying a microgravitational force at the outlet end of said separation channel to induce hydrodynamic flow of the sample solution containing isoelectrically focused analyte zones along the separation channel and to mobilize and further focus of the analyte zones; and (f) whole column image detection means for monitoring the focusing of analytes into zones in real time.
Date Recue/Date Received 2021-06-30
(a) a horizontal capillary having a central separation channel portion, an integral inlet portion at one end thereof and an integral outlet channel portion at the other end thereof;
(b) at each end of said separation channel portion, an electrolyte tank including an ion conductive barrier separating fluid inside the tank from fluid in the separation column;
(c) means for filling the capillary with a sample of analyte solution comprising autosampler means, a reservoir for sample solution and switch valve means for selectively connecting said autosampler and said inlet reservoir to said inlet channel;
(d) means for establishing isoelectric focusing of analytes within said separation channel into zones comprising an electrode in each of said electrolyte tanks and a switchable high voltage supply connected across the electrodes immersed in the electrolyte in said tanks;
(e) means for applying a microgravitational force at the outlet end of said separation channel to induce hydrodynamic flow of the sample solution containing isoelectrically focused analyte zones along the separation channel and to mobilize and further focus of the analyte zones; and (f) whole column image detection means for monitoring the focusing of analytes into zones in real time.
Date Recue/Date Received 2021-06-30
2. Apparatus according to claim 1, wherein said means for applying a micro-gravitational force at the outlet end of said separation channel comprises an outlet reservoir for sample solution, a flexible tube having one end immersed in fluid in said outlet reservoir and the other end connected to said outlet channel by a hydrodynamic flow control valve, and means for the controlled vertical raising or lowering of said outlet valve, thereby to vary the hydrostatic pressure and establish said microgravitational force for inducing hydrodynamic flow of the sample solution along the separation channel.
3. Apparatus according to claim 2, wherein said whole column imaging detection means comprises lamp means for emitting a generally planar beam of light from below the separation channel along the entire length thereof, and a CCD
imaging camera focused above the channel to detect light passing through or emitted from the entire length of the separation channel.
imaging camera focused above the channel to detect light passing through or emitted from the entire length of the separation channel.
4. Apparatus according to claim 2 or claim 3, further comprising means for effecting chemical mobilization and further focusing of the isoelectrically focused analyte zones within the separation channel.
5. Apparatus according to claim 4, wherein said means for effecting chemical mobilization comprises a third electrolyte tank containing a suitable solution, said tank including an ion conductive barrier separating the fluid therein from fluid in the separation column, an electrode extending into said chemical mobilization tank, and switching means for completing a circuit with said high voltage supply and one or the other of the two electrolyte tanks disposed at the ends of said separation channel portion.
6. A method, comprising:
introducing a sample into a separation channel, the sample including a mixture of proteins, carrier ampholytes and a sieving solution;
establishing an electric field across the separation channel;
separating the proteins into zones based on a value associated with the pl of Date Recue/Date Received 2021-06-30 the proteins; and continuously monitoring the separating of the proteins using an optical device.
introducing a sample into a separation channel, the sample including a mixture of proteins, carrier ampholytes and a sieving solution;
establishing an electric field across the separation channel;
separating the proteins into zones based on a value associated with the pl of Date Recue/Date Received 2021-06-30 the proteins; and continuously monitoring the separating of the proteins using an optical device.
7. The method of claim 6, further comprising:
after the separating, inducing a hydrodynamic flow in the separation channel;
introducing a second volume of the sample into the separation channel in response to the hydrodynamic flow;
separating ampholytes from the second volume of the sample into the zones based on a value associated with the pl of the ampholytes; and continuously monitoring the separating of the ampholytes using an optical device.
after the separating, inducing a hydrodynamic flow in the separation channel;
introducing a second volume of the sample into the separation channel in response to the hydrodynamic flow;
separating ampholytes from the second volume of the sample into the zones based on a value associated with the pl of the ampholytes; and continuously monitoring the separating of the ampholytes using an optical device.
8. The method of claim 6, wherein the monitoring includes monitoring along substantially an entire length of the separation channel simultaneously.
9. The method of claim 6, wherein the sieving solution is methyl cellulose.
Date Recue/Date Received 2021-06-30
Date Recue/Date Received 2021-06-30
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA3123719A CA3123719A1 (en) | 2010-08-05 | 2010-08-05 | Method to perform limited two dimensional separation of proteins and other biologicals |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA3123719A CA3123719A1 (en) | 2010-08-05 | 2010-08-05 | Method to perform limited two dimensional separation of proteins and other biologicals |
| CA2712213A CA2712213C (en) | 2010-08-05 | 2010-08-05 | Method to perform limited two dimensional separation of proteins and other biologicals |
Related Parent Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA2712213A Division CA2712213C (en) | 2010-08-05 | 2010-08-05 | Method to perform limited two dimensional separation of proteins and other biologicals |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA3123719A1 true CA3123719A1 (en) | 2012-02-05 |
Family
ID=45566691
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA3123719A Pending CA3123719A1 (en) | 2010-08-05 | 2010-08-05 | Method to perform limited two dimensional separation of proteins and other biologicals |
| CA2712213A Active CA2712213C (en) | 2010-08-05 | 2010-08-05 | Method to perform limited two dimensional separation of proteins and other biologicals |
Family Applications After (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA2712213A Active CA2712213C (en) | 2010-08-05 | 2010-08-05 | Method to perform limited two dimensional separation of proteins and other biologicals |
Country Status (1)
| Country | Link |
|---|---|
| CA (2) | CA3123719A1 (en) |
-
2010
- 2010-08-05 CA CA3123719A patent/CA3123719A1/en active Pending
- 2010-08-05 CA CA2712213A patent/CA2712213C/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CA2712213A1 (en) | 2012-02-05 |
| CA2712213C (en) | 2021-08-24 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US10107782B2 (en) | Method to perform limited two dimensional separation of proteins and other biologicals | |
| CA2657317C (en) | Method and apparatus for precise selection and extraction of a focused component in isoelectric focusing performed in micro-channels | |
| US10866246B2 (en) | Devices, methods and kits for sample characterization | |
| JP6422131B2 (en) | Capillary device for separation analysis, microfluidic chip for separation analysis, protein or peptide analysis method, electrophoresis apparatus, and microfluidic chip electrophoresis apparatus for separation analysis | |
| US5395502A (en) | Apparatus for performing and universally detecting capillary isoelectric focusing without mobilization using concentration gradient imaging systems | |
| US11285484B2 (en) | Multichannel isoelectric focusing devices and high voltage power supplies | |
| JP3410099B2 (en) | Isoelectric focusing method and apparatus using no carrier ampholyte | |
| EP2773959B1 (en) | Protein fractionation based on isoelectric focusing | |
| Bergmann et al. | Potential of on-line isotachophoresis-capillary zone electrophoresis with hydrodynamic counterflow in the analysis of various basic proteins and recombinant human interleukin-3 | |
| US20230381781A1 (en) | Isoelectric focusing devices and fixtures | |
| CA2712213C (en) | Method to perform limited two dimensional separation of proteins and other biologicals | |
| Wu et al. | Analysis of proteins by CE, CIEF, and microfluidic devices with whole-column-imaging detection | |
| Mikuš et al. | Column coupling electrophoresis in biomedical analysis | |
| JP2008309539A (en) | Two-dimensional electrophoretic apparatus | |
| EP0621947B1 (en) | System for performing and universally detecting capillary isoelectric focusing without mobilization using concentration gradient imaging systems | |
| Végvári | Peptide and protein separations by capillary electrophoresis and electrochromatography | |
| Kilár | Capillary Isoelectric Focusing | |
| Zhan | Development of Isoelectric Focusing Techniques for Protein Analyses |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| EEER | Examination request |
Effective date: 20210630 |
|
| EEER | Examination request |
Effective date: 20210630 |
|
| EEER | Examination request |
Effective date: 20210630 |
|
| EEER | Examination request |
Effective date: 20210630 |
|
| EEER | Examination request |
Effective date: 20210630 |
|
| EEER | Examination request |
Effective date: 20210630 |
|
| EEER | Examination request |
Effective date: 20210630 |
|
| EEER | Examination request |
Effective date: 20210630 |
|
| EEER | Examination request |
Effective date: 20210630 |