Electrophoretic Separation Element with Trapping for Protein Resolution
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current protein separation techniques, such as electrophoretic methods, face limitations in efficiently separating complex protein mixtures, particularly in achieving precise isoelectric focusing and size-based separation, especially when dealing with samples that require high resolution and minimal sample quantities.
Innovation Solution
The development of devices with a fluid flow path and electrophoretic separation elements that apply an electric field and include trapping elements, allowing for precise pH control and orthogonal separation mechanisms, enabling simultaneous or sequential movement of samples past separation elements to trap and separate proteins based on isoelectric point and size.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If traditional electrophoretic techniques (IEF and SDS-PAGE) are used for protein separation, then proteins can be separated based on isoelectric point and size, but the separation resolution is insufficient for complex protein mixtures and requires large sample quantities
Solution Approach 1:
The patent implements two-dimensional separation by combining isoelectric focusing (separation by charge/pH) in the first dimension with size-based gel electrophoresis in the second dimension. This orthogonal approach separates proteins along two independent parameters simultaneously, achieving much higher resolution for complex mixtures than single-dimensional methods, while requiring minimal sample quantities due to the cumulative separation effect.
2Measurement precision
If complex mixtures of ampholytes are used to create pH gradients for IEF, then proteins can be separated by isoelectric point, but the device complexity and chemical requirements increase significantly
Solution Approach 1:
The patent controls pH gradients by adjusting electrical parameters (voltage, current, pulse duration) and temporal parameters (sequential vs. simultaneous application) rather than relying solely on complex chemical ampholyte mixtures. By dynamically controlling the electric field parameters, the system achieves precise isoelectric focusing with simplified chemical requirements.
3Measurement precision
If proteins are separated sequentially through multiple electrophoretic steps, then high resolution separation is achieved, but the time required for complete separation increases
Solution Approach 1:
The patent performs isoelectric focusing in the first dimension before applying the second dimension gel electrophoresis separation. This preliminary separation by charge simplifies the subsequent size-based separation, allowing both dimensions to work efficiently together. The sequential preparation and separation steps are optimized to minimize total time while maintaining high resolution.
Solution Approach 2:
The system maintains continuous separation action by implementing both electrophoretic dimensions in an integrated flow system where proteins continuously migrate through both separation mechanisms without interruption or intermediate handling, reducing total separation time while preserving resolution.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This approach allows for efficient, high-resolution separation of proteins into distinct fractions, enabling precise isoelectric focusing and size separation, even with small sample quantities, and facilitates further processing and analysis, such as 2-dimensional separation protocols.
Implementation Method 1
Proteins, in common with the majority of biomolecules, are charged or can be made to be charged by defining the media in which they are present. Consequently they will move, in solution, under the influence of an electric field with a velocity which is dependent on the charge to mass ratio of the protein
Implementation Method 2
IEF separates proteins on the basis of their isoelectric point. When proteins are introduced into this system, their charge will depend on the pH of the environment in which they find themselves. If the environmental pH is lower than the protein's pI, then it will have a net positive charge and will migrate towards the negative electrode
Implementation Method 3
In these separation protocols, proteins may be separated by causing them to migrate through a sieving structure, such as one produced by making a cross-linked gel or a solution of entangled polymers. In such applications, small protein molecules will travel faster than large protein molecules and any mixture of these will therefore separate according to molecular weight
Data Source
AI summary
Devices and methods for separating sample constituents are provided. The subject devices are characterized by having a fluid flow path with at least one electrophoretic separation element positioned at a region thereof. The separation element includes an element for applying an electric field across the fluid flow path and a trapping element for trapping sample constituents that migrate out of the flow path when an electric field is applied across the fluid flow path. In using the subject devices, sample is moved past the separation element and an electric field is applied across the flow path such that constituents of the sample migrate into the trapping element. The subject devices and methods find use in a variety of applications, including protein separation applications.