Isoelectric point (pI) is the pH value at which the molecule carries no net electrical charges, which is one of important characters for zwitterionic molecules such as aimno acids, peptides, and proteins. Since pI can be a critical factor to affect the overall pharmacokinetic (PK) behavior, it is necessary to assess pI during drug development.
Isoelectrofocusing (IEF) is the most common and widely used electrophoretic technique to determine pI of a protein or separate proteins depending on their pI s. The net charges of proteins are determined by the overall pH of proteins' environment. When the acidic groups in a protein are more than the number of basic groups, the protein's pI is at a low pH value. In the contrary, the protein's pI is at a high pH value, if the number of basic groups exceeds the number of acidic groups of the protein. During electrophoresis, positively charged proteins, which are at pH values below the pI, will move toward the cathode on pH-gradient gel. Whereas, negatively charged proteins with pH values above the pI will migrate to the anode. Besides, protein at its pI has no net charge, and will not move during electrophoresis.
In IEF, proteins migrate to their respective pI and remain stabilized. To conduct successful IEF, stable and linear pH gradients are important. Creative Proteomics provides a high-resolution IEF capable of separating proteins with different pIs. Depending on your samples, different antibodies, antigens, and enzymes will be employed to ensure the successful fractionation. Buffers and ampholyte conditions will also be optimized to separate proteins across a broad range of pH.
Advantages of our Isoelectric point determination with IEF:
- Rich experience in pI determination.
- High sensitivity and accuracy to detect proteins with different post-translational modifications.
- Rapid turnaround time to provide comprehensive report.
- Customized service: according to you sample type and sample size, optimized protocols will be deployed.
We provide but are not limited to:
- Isoelectric point determination of proteins
- Separation of proteins and peptides
Methods of Isoelectric Point Determination with IEF
A vertical, one-dimensional, IEF protocol will be conducted to determine pI of your samples. Compared to immobilized pH gradient (IPG) strips, the one-dimensional vertical IEF is simpler and less time-consuming, and can analyze multiple samples at the same time.
After the samples are prepared with optimal buffers, samples and standard marker will be applied onto the IEF gel with gradient pH to perform electrophoresis. When the electrophoresis is completed, proteins will be transferred to a solid support, such as PVDF or nitrocellulose filter membrane. Then the immunodetection procedures will be carried out to detect proteins/peptides, and determine pIs.
Creative Proteomics's experienced scientists will optimize protocols to provide accurate pI determination of different types of samples, and customized services will be provided to help customers solve analytical and technical problems.
More than isoelectric point determination with IEF.
Want to learn more about isoelectric point determination with IEF? We provide one-stop analysis of isoelectric point determination.
References
- Ganapathy-Kanniappan S. (2019). pI Dtermination of Native Proteins In Biological Samples. Current Protocols in Protein Science, e85. doi: 10.1002/cpps.85.
- George Komis and Jozef Šamaj (eds.), Plant MAP Kinases: Methods and Protocols, Methods in Molecular Biology, vol. 1171, doi 10.1007/978-1-4939-0922-3_4.
