Isoelectric Focusing (IEF) Technology

Isoelectric focusing (IEF) plays a vital role in the separation and purification of proteins and peptides during biopharmaceutical development. CD Formulation integrates cutting-edge IEF technology into our protein/peptide characterization technology platforms to provide precise insights into your biopharmaceutical development plan. With our IEF technology, you can obtain key information about protein properties to support formulation development, purification process development, product stability assessment, and release testing in quality control (QC).

What is Isoelectric Focusing (IEF)?

IEF, an effective protein electrophoresis analysis method, is one of the most commonly used techniques for separating proteins or peptides. This technique takes advantage of the fact that proteins have different isoelectric points, at which they have no net charge and will not migrate in the electric field. Currently, IEF technology is able to distinguish biomolecules with isoelectric points that differ by only 0.001 pH units. This technology is widely used in the fields of biochemistry, molecular biology, and clinical medical research with the characteristics of high resolution, good reproducibility, large sample capacity, and simple and fast operation. It is a separation technology that is very suitable for analytical and preparative applications.

Isoelectric Focusing (IEF) Principle

IEF separation is based on the pH dependence of the electrophoretic mobility of protein molecules. This technology uses the difference in the isoelectric point of proteins or polypeptides to effectively separate and detect proteins or polypeptides by establishing a stable, continuous, and linear pH gradient in polyacrylamide or agarose gel. Since proteins are amphoteric compounds, their charge is related to the pH value of the medium. Proteins with a net charge migrate in the opposite polarity direction during electrophoresis (pH>pI, proteins release protons, carry negative charges, and move toward the anode; pH=pI, proteins are uncharged and stop moving; pH<pI, proteins carry positive charges and move toward the cathode). When the protein reaches the pI position in the gel, the current reaches a minimum and the protein stops moving.

According to different methods of establishing the pH gradient, it is divided into carrier ampholyte pH gradient-IEF (CAPG-IEF) and immobilized pH gradient-IEF (IPG-IEF).

• CAPG-IEF: When carrier ampholyte components are added to a certain medium, a pH gradient is gradually formed from the anode to the cathode after applying a voltage.
• IPG-IEF: The use of immobilized pH gradient gel avoids the problems of long focusing time, unstable pH gradient, cathode drift, etc., which are caused by carrier ampholytes.

Fig. 1 Principle of isoelectric focusing. (Pergande MR, et al., 2017)

Our Services Related to Isoelectric Focusing (IEF)

Thanks to decades of experience in supporting protein/peptide biopharmaceutical development and manufacturing using IEF technology, our team of highly qualified experts offers a range of IEF-related services to accelerate the implementation and success of your projects.

Our scientists will work with you to continuously optimize IEF programs to support all stages of protein/peptide drug development and manufacturing - from early protein/peptide drug discovery to late-stage drug development and post-release testing under GMP.

Utilizing cutting-edge IEF technology, we support the following protein/peptide characterization plans, including but not limited to:

Protein Purification Analysis

IEF is often used as the first step in multidimensional protein separation approaches. Our scientists separate proteins based on their isoelectric points to screen and design the best downstream purification process for you.

Protein Charge Variants Characterization

Proteins can exist in multiple charged forms (isomers) that may differ in post-translational modifications such as phosphorylation or glycosylation. IEF can analyze these charge variants, providing valuable insights into protein function and regulation.

Protein Isoelectric Point (pI) Determination

By performing IEF, our scientists can accurately determine the isoelectric point of a protein. This information is critical for predicting protein behavior at different pH environments and for designing buffer systems in a variety of biochemical applications.

Protein Stability and Conformation Assessment

The changes in a protein's isomeric profile under different conditions (e.g., temperature, pH, denaturants) can provide insight into its stability and conformational state. Our scientists use IEF to assess the stability of a protein under different conditions such as pH and ionic strength. The changes in pI under different pressure conditions are analyzed to infer protein stability and folding.

Quality Control

IEF is critical for the quality control of protein-based therapeutics. Ensuring consistent charge variant profiles between batches guarantees product safety and efficacy, making IEF an essential component of the protein and peptide manufacturing process

Protein Identification

IEF is often used as part of two-dimensional gel electrophoresis (2DE) followed by SDS-PAGE. This combination allows for a more detailed analysis of complex protein mixtures, enabling the identification and comparison of proteins based on their pI and molecular weight.

Protein Quantification

In addition to qualitative analysis, IEF can be used to quantify protein abundance. Our scientists estimate the relative concentration of a protein in a sample by comparing the intensity of the focused protein band to a standard.

Our IEF Equipment

IEF is a technique with extremely high resolution and sensitivity. When used with conventional protein stains, the technique is able to detect proteins down to about 100 mg/L. When used in conjunction with blotting and probing techniques, the technique can detect proteins down to less than 1 mg/L. To support more thorough protein and peptide characterization analysis services, our lab is equipped with a range of IEF combination equipment to support your project needs.

IEF-Polyacrylamide Gel Electrophoresis (IEF-PAGE)

IEF is often used in conjunction with gel electrophoresis equipment PAGE for protein separation and analysis. IEF is a high-resolution separation technique that can separate individual proteins by their isoelectric point (pI), while gel electrophoresis can be separated by molecular weight. Combining these two techniques allows for a higher level of protein separation and analysis, and is commonly seen in proteomics studies and sample preparation.

IEF-Mass Spectrometry (IEF-MS)

When IEF is combined with MS, it can identify proteins and their isoforms with high sensitivity. Proteins can be focused in IEF and subsequently analyzed by MS to obtain qualitative and quantitative insights, including molecular weight, amino acid sequence information, and structural characteristics of the protein.

Fig.2 Examples of isoelectric focusing (IEF) methods incorporated prior to mass spectrometry (MS) detection. (Pergande MR, et al., 2017)

Types of IEF Available Types of IEF Analysis

Capillary Isoelectric Focusing (CIEF)

CIEF is an advanced analytical technique that combines the principles of isoelectric focusing and capillary electrophoresis. Charged biomolecules can be separated with high resolution and efficiency in narrow capillaries (25-75 μM inner diameter). The workflow can be easily automated in the laboratory.

IEF-Gel Electrophoresis

IEF-gel electrophoresis, a widely used technique in IEF, is a powerful technique for separating proteins based on their isoelectric point (pI). The technique exploits the fact that proteins migrate in a pH gradient until they reach a point where their net charge is zero, effectively achieving separation based on charge properties alone. It effectively reduces convection and, combined with the gel sieving effect, achieves precise separation of proteins based on size.

Microfluidic Chip-Based IEF

This technology is an advanced laboratory technique that combines microfluidics with IEF. It has many advantages over capillary electrophoresis, including lower sample consumption, higher resolution, faster analysis time, and greater automation and integration.

Advantages of Our Isoelectric Focusing (IEF)

• High resolution allows for a more complete separation of solutes.
• Highly sensitive detection allows for the determination of low-abundance proteins.
• Wide pH range allows for separating a variety of proteins with different charge characteristics.
• Combined with a variety of staining techniques, allows visualization of proteins for qualitative and quantitative analysis.
• It can be performed under conditions that preserve the native state of proteins.
• Minimal sample consumption.

Custom Isoelectric Focusing (IEF) Services

Why Choose Our Isoelectric Focusing (IEF) Technology?

• We have a team of experts with extensive experience in IEF analytical method development and validation, and are able to provide customers with accurate analysis and effective advice.
• We have accumulated decades of expertise and successful project experience using IEF technology to support the development of protein/peptide biopharmaceuticals.
• We use state-of-the-art IEF instruments and cutting-edge methods to optimize the quality and reproducibility of protein separation processes, resulting in precise and accurate results.
• All of our services are based on comprehensive data collection and in-depth analysis to help customers make informed decisions.
• Our services can be customized to your specific needs, whether focused on protein characterization, purification or analysis.
• Our services include not only the technical execution of IEF, but also comprehensive consulting and support throughout the project, helping you interpret the results and integrate the findings into broader research or clinical goals.

Publication

CD Formulation aims to provide a powerful analytical tool for the separation, purification, and characterization of proteins and peptides. Please feel free to contact us if you are interested in our services. Learn how our IEF technology can support the smooth implementation of your protein/peptide biopharmaceutical program.

How It Works

STEP 1

Submit a service request describing your specific research or development need.

STEP 2

We'll email you to provide your quote and confirm order details if applicable.

STEP 3

Execute the project with real-time communication, and deliver the final report promptly.