Difference Between Chromatofocusing and Isoelectric Focusing

The key difference between chromatofocusing and isoelectric focusing is that chromatofocusing is a type of column chromatography method which utilizes ion exchange resins while isoelectric focusing is a type of electrophoresis technique which utilizes immobilized pH gradient gels.

1. What is the principle of isoelectric focusing?
2. What is isoelectric focusing used for?
3. What is capillary isoelectric focusing?
4. What is the purpose of using Ampholytes in isoelectric focusing?
5. What is meant by isoelectric point?
6. Who discovered isoelectric focusing?
7. What Cannot be a reason for using electrophoresis?
8. What affects isoelectric point?
9. What is Pharmalyte?
10. What is the force that is used to move charged species in the method of isoelectric focusing?
11. How would you separate proteins according to their isoelectric point?

What is the principle of isoelectric focusing?

Isoelectric Focusing or IEF is a method of separating proteins according to their Isoelectric points in a pH gradient. Isoelectric point denoted as pI is defined as the pH at which protein carry no net charge, or pH at which protein become immobile in an electric field.

What is isoelectric focusing used for?

IEF is used mainly to separate proteins for analysis or purification. It measures the isoelectric points (pI) of proteins and uses the unique pI values of proteins to purify them. The pI of any particular protein is defined as the specific pH at which it carries no net electrical charge.

What is capillary isoelectric focusing?

Capillary isoelectric focusing (cIEF) is a high-resolution analytical technique that allows the separation of protein/peptide mixtures, protein glycoforms and other charge variants, based on their isoelectric point (pI).

What is the purpose of using Ampholytes in isoelectric focusing?

The sample is usually combined with carrier ampholytes to assist in migration. Ampholytes are a mixture of charged molecules with a range of pIs that matches the pI range of the IPG strip. The migration of the ampholytes encourages the sample molecules to move along the pH gradient.

What is meant by isoelectric point?

The isoelectric point (pI) is the pH at which a particular molecule carries no net electrical charge. The net charge on the molecule is affected by the pH of its surrounding environment and can become more positive or negative due to the gain or loss of protons, respectively.

Who discovered isoelectric focusing?

It is usually the method of choice for the preparation of isoforms of proteins. An important development of the basic polyacrylamide gel IEF procedure described by O'Farrel in 1975 is the concept of two-dimensional electrophoresis.

What Cannot be a reason for using electrophoresis?

9. When is electrophoresis not used? Explanation: Electrophoresis cannot be used in separation of lipids.

What affects isoelectric point?

The standard nomenclature to represent the isoelectric point is pH(I). ... The net charge on the molecule is affected by pH of its surrounding environment and can become more positively or negatively charged due to the gain or loss, respectively, of protons (H⁺).

What is Pharmalyte?

Pharmalyte. ® IEF carrier ampholytes are mixtures of. 600-700 different amphoteric compounds within a defined isoelectric point range. Pharmalyte products are synthesized by the co-polymerization of glycine, glycylglycine, various amines and epichlorhydrin.

What is the force that is used to move charged species in the method of isoelectric focusing?

The driving force for the proteins is the electric force. Recall that each protein has an isoelectric point; when the protein's environmental pH equals the isoelectric point, the protein will become neutral and stop migrating (it loses the electric force). The physiological pH is about 7.4.

How would you separate proteins according to their isoelectric point?

A protein can be purified according to its protein isoelectric point by running the said protein through an ion exchange column or a pH-graded gel. At the isoelectric point, a protein has no net charge. Above the isoelectric point, a protein carries a net negative charge—below it, a net positive charge.