Isoelectric point (pI) is the pH value at which the overall net charge of a macromolecule equals zero. pI is one of important physico-chemical parameters to influence overall pharmacokinetic behavior, so that it's necessary to assess pI during drug development.
pI can be determined by various experiments, including isoelectric focusing experiments, free flow electrophoresis, capillary electrophoresis, and in-gel electrophoresis experiments using immobilized pH gradient (IPG) strips. Amphoteric compounds are fractionated depending on their pI along a continuous pH gradient in Isoelectric focusing (IEF), which is the most common used electrophoretic technique to determine pI. And capillary IEF (cIEF) is the technique that can quantitatively and experimentally analyze the pI, and is widely adopted in most chemistry laboratories. Two ways to perform cIEF: coated or uncoated capillaries. In terms of using coated capillary, a two-step protocol is required due to the suppression of electroendoosotic flow (EOF), including a focusing step, and mobilization. As for uncoated cIEF, focusing happens when the entire pH gradient and train of bands is migrating to the capillary outlet. Since the coated cIEF has higher reproducibility and reduced chance of accidental protein absorption to the silica wall, it is more preferable and commonly used.
Determination of pI with precision is not an easy task, since a variety of experimental parameters affect the value, such as temperature, salt, solubilizers, calibration, etc. Our experienced scientific team will offer sensitive, accurate and reliable pI determination with cIEF for different types of samples.
Advantages of our Isoelectric point determination with cIEF:
- 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 cIEF
In cIEF, pI markers are the only way for charting the course of pH gradients. Hence, employment of precise isoelectric point makers for cIEF is also critical. A set of 16 synthetic oligopeptides (trimers to hexamers), which have sharp focusing, stability, high purity and high solubility, will be used as isoelectric point makers to determine pI value. Each peptide in this set is made to contain one Trp residue for UV absorption detection and other residues with ionic side-chains for sharply-focusing peak during cIEF.
The samples will be first mixed with ampholytes, stablilizers and pI markers, and loaded to the capillary. One end of the capillary filled with sample mixture then will be submerged in anolyte and the other in catholyte. After a voltage is applied to the system, pH gradient forms during focusing through the introduction of hydronium ions from the anolyte and hydroxyl ions from the catholyte, and ampholytes and protein samples focus before the detection window.
Experts from Creative Proteomics will optimize protocols and pI markers to provide precise 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 cIEF.
Want to learn more about isoelectric point determination with cIEF? We provide one-stop analysis of isoelectric point determination.
References
- Righetti P.G. Determination of the isoelectric point of proteins by capillary isoelectric focusing. J. Chromatogr. A 1037 (2004) 491–499.
- Audain E., Ramos Y., Hermjakob H., et al., Accurate estimation of isoelectric point of protein and peptide based on amino acid sequences. Bioinformatics, 32(6), 2016, 821–827.
