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.
Chromatofocusing and isoelectric focusing are two techniques that separate proteins according to their isoelectric points. Both chromatofocusing and isoelectric focusing have great resolving power. Chromatofocusing is a high-resolution column chromatography method while isoelectric focusing is an electrophoretic separation method. Moreover, chromatofocusing does not use an electric field while isoelectric focusing needs an electric field.
CONTENTS
1. Overview and Key Difference
2. What is Chromatofocusing
3. What is Isoelectric Focusing
4. Similarities Between Chromatofocusing and Isoelectric Focusing
5. Side by Side Comparison – Chromatofocusing vs Isoelectric Focusing in Tabular Form
6. Summary
What is Chromatofocusing?
Chromatofocusing is a type of high elution chromatography that separates proteins according to differences in their isoelectric point. It uses an ion exchange resin column and an internally developed pH gradient. Unlike isoelectric focusing, chromatofocusing does not involve an electric field. Chromatofocusing is a powerful purification technique which can resolve highly similar molecules even only differing by 0.02 pH units. Therefore, chromatofocusing is best suited as a final polishing step when high resolution is required in separating very similar components.
In chromatofocusing, the sample is applied to the column by mixing it with the start buffer. Proteins that are at a pH above their isoelectric point are negatively charged and they remain near the top of the column while other proteins which are at the pH below their isoelectric point, migrate down the column. The protein with the highest isoelectric point is eluted first while the protein with the lowest isoelectric point is eluted last from the column. This technique is affected by several factors such as protein precipitation and linearity of the pH gradient.
What is Isoelectric Focusing?
Isoelectric focusing is a technique that separates different molecules based on their isoelectric point. It is also known as electrofocusing. This technique is often used for protein separation in a gel. In isoelectric focusing, the sample is added into immobilized pH gradient (IPG) gels. IPG gel is an acrylamide gel matrix copolymerized with a pH gradient. The proteins migrate towards the cathode until they find the pH region of their isoelectric points. When a protein reaches the pH which has no net charge, migration ceases and gives a stationary band. Likewise, each protein gives a band at a point in the pH gradient corresponding to its isoelectric point.
Figure 01: Isoelectric Focusing
Isoelectric focusing is the first step of two-dimensional gel electrophoresis. In that technique, proteins are first separated according to their isoelectric points by isoelectric focusing and then separated by their molecular weight through SDS-PAGE.
What are the Similarities Between Chromatofocusing and Isoelectric Focusing?
- Chromatofocusing and isoelectric focusing are two highly resolving methods.
- Both are based on the isoelectric point of proteins.
- These techniques use a pH gradient.
- Both procedures are informative for the characterization of human haemoglobin variants.
- They can resolve molecules where pI values differ by as little as 0.02 pH units.
What is the Difference Between Chromatofocusing and Isoelectric Focusing?
Chromatofocusing is a high-resolution column chromatography technique that separates proteins according to their isoelectric point. In contrast, isoelectric focusing is an electrophoretic technique which separates proteins according to their isoelectric point by the application of an electric field along a pH gradient formed in a capillary. So, this is the key difference between chromatofocusing and isoelectric focusing.
Another difference between chromatofocusing and isoelectric focusing is that chromatofocusing does not involve an electric field while isoelectric focusing needs an electric field.
Summary – Chromatofocusing vs Isoelectric Focusing
Chromatofocusing and isoelectric focusing are two similar techniques which have high resolving ability of molecules, especially very similar molecules. Chromatofocusing is a high-resolution column chromatography technique while isoelectric focusing is an electrophoresis method. Chromatofocusing does not involve an electric field, unlike isoelectric focusing. Thus, this summarizes the difference between chromatofocusing and isoelectric focusing.