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US 10,107,782 B2Granted
Method to perform limited two dimensional separation of proteins and other biologicals
AssigneeProteinSimple
InventorsTiemin Huang, Jiaqi Wu
Life of this patent
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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.
Claims20
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.
7 more elements
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.
5 more elements
Dependencies are inferred from the claim text.