Method to perform limited two dimensional separation of proteins and other biologicals

Patent metadata

Field	Value
Publication number	US-10107782-B2
Application number	US-35872409-A
Country	US
Kind code	B2
Filing date	Jan 23, 2009
Priority date	Jan 25, 2008
Publication date	Oct 23, 2018
Grant date	Oct 23, 2018

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.

First claim

We claim:

1. A method, comprising:
loading an inlet reservoir and a separation channel of an electrophoretic cell with a solution including a plurality of proteins, an amphoteric carrier medium, and a sieving solution, the inlet reservoir fluidically coupled to the separation channel;
during a first separation stage:
establishing a pH gradient across the separation channel, the pH gradient being static during the first separation stage;
applying a voltage to the separation channel in the presence of the pH gradient such that the plurality of proteins are focused into a zone having a pH value corresponding to a pI value of each protein from the plurality of proteins in that zone, the zone being static during the first separation stage;

during a second separation stage mutually exclusive from the first separation stage:
drawing the sample from the inlet reservoir into the separation channel inducing a hydrodynamic flow within the separation channel causing the zone to move away from the inlet reservoir such that each protein from the plurality of proteins is displaced from the pH value corresponding to the pI value of each protein from the plurality of proteins and such that each protein from the plurality of proteins moves towards the zone effecting a separation of the plurality of proteins by mobility; and

continuously monitoring the plurality of proteins during the second separation stage using a whole column imaging detector; and
detecting a separation of a first protein from a second protein during the second separation stage due to the first protein having a different mobility than the second protein, the first protein and the second protein each being from the plurality of proteins.

2. The method of claim 1 , wherein the plurality of proteins are a first plurality of proteins, the pI value is a first pI value, and the sample includes a second plurality of proteins having a plurality of pI values different from the first pI value.

3. The method of claim 1 wherein the first separation stage concludes when the plurality of proteins are focused into the zone.

4. The method of claim 1 , further comprising:
continuously monitoring the plurality of proteins during the first separation stage using the whole column imaging detector, the hydrodynamic flow induced in response to the whole column imaging detector detecting that each protein from the plurality of proteins is focused into the zone.

5. The method of claim 1 , wherein the first protein has a first mobility and the second protein has a second mobility greater than the first mobility such that the second protein moves through the sieving solution towards the zone during the second separation stage faster than the first protein.

6. The method of claim 1 , wherein the plurality of proteins are focused during the first separation stage via isoelectric focusing.

7. The method of claim 1 , wherein the monitoring includes monitoring along substantially an entire length of the separation channel simultaneously.

8. The method of claim 1 , wherein the sieving solution is methyl cellulose.

9. The method of claim 1 , wherein:
the plurality of proteins is a first plurality of proteins;
the zone is a first zone having a first pH value corresponding to a first pI value of each protein from the first plurality of proteins;
the solution includes a second plurality of proteins;
during the first separation stage the voltage is applied to the separation channel such that a first plurality of protein are focused into the first zone and such that a second plurality of proteins are focused into a second zone having a second pH value corresponding to a second pI value of each protein from the second plurality of proteins; and
the first zone and the second zone are static during the first separation stage.

10. The method of claim 1 , wherein:
the plurality of proteins is a first plurality of proteins;
the zone is a first zone having a first pH value corresponding to a first pI value of each protein from the first plurality of proteins;
the solution includes a second plurality of proteins;
during the first separation stage the voltage is applied to the separation channel such that a first plurality of protein are focused into the first zone and such that a second plurality of proteins are focused into a second zone having a second pH value corresponding to a second pI value of each protein from the second plurality of proteins;
during the second separation stage the hydrodynamic flow causes the first zone to move away from the inlet reservoir such that each protein from the first plurality of proteins is displaced from the first pH value corresponding to the first pI value such that each protein from the first plurality of proteins moves towards the first zone effecting a separation of the first plurality of proteins by mobility; and
during the second separation stage the hydrodynamic flow causes the second zone to move away from the inlet reservoir such that each protein from the second plurality of proteins is displaced from the second pH value corresponding to the second pI value such that each protein from the second plurality of proteins moves towards the second zone effecting a separation of the second plurality of proteins by mobility.

11. A method, comprising:
loading a separation channel having a first end and a second end with a solution including a plurality of proteins, an amphoteric carrier medium, and a sieving solution;
during a first separation stage:
ionically coupling a first anolyte reservoir containing an acid to the first end of the separation channel and a first catholyte reservoir containing a base to the second end of the separation channel such that a first pH gradient is established across a length of the separation channel, the first pH gradient being static during the first separation stage;
applying a voltage to the separation channel in the presence of the first pH gradient such that the plurality of proteins are focused into a zone having a pH value corresponding to a pI value of each protein from the plurality of proteins in that zone, the zone being static during the first separation stage;

during a second separation stage mutually exclusive from the first separation stage:
ionically coupling at least one of a second anolyte reservoir different from the first anolyte reservoir to the first end of the separation channel or a second catholyte reservoir different from the first catholyte reservoir to a second end of the separation channel, such that the at least one of the second analyte reservoir or the second catholyte reservoir replaces at least one of the first analyte reservoir or the first catholyte reservoir such that a second pH gradient different from the first pH gradient is established across the length of the separation channel and such that each protein from the plurality of proteins is displaced from the pH value corresponding to the pI value of each protein from the plurality of proteins and such that each protein from the plurality of proteins moves towards the zone effecting a separation of the plurality of proteins by mobility; and
continuously monitoring the plurality of proteins during the second separation stage using a whole column imaging detector.

12. The method of claim 11 , wherein each protein from the plurality of proteins has the pI value.

13. The method of claim 11 , wherein the plurality of proteins are a first plurality of proteins, the sample includes a second plurality of proteins.

14. The method of claim 11 , wherein the second anolyte reservoir contains a non-acid.

15. The method of claim

Assignees

• Huang Tiemin
• Wu Jiaqi
• ProteinSimple

Inventors

• Huang Tiemin
• Wu Jiaqi

Classifications

• G01N27/44773

  Multi-stage electrophoresis, e.g. two-dimensional electrophoresis · CPC title

• C07K1/285

  multi dimensional electrophoresis · CPC title

• G01N27/44795 Primary

  Isoelectric focusing · CPC title

• G01N27/44721

  by optical means · CPC title

• C07K1/36

  by a combination of two or more processes of different types · CPC title