advertisement  |
The First Dimension: Choosing The Right Equipment For Isoelectric Focusing (IEF)
Buying Tips
Feb 25 '04
 |
|
|
 |
Introduction |
|
|
 With the heightened interest in proteomics-based studies, there has been an increased need for robust and reproducible protein fractionation methods. Given the almost unmanageable complexity of the set of proteins characteristic of a given cell type or tissue, the interest in multidimensional fractionation schemes has also seen an upsurge. By multidimensional, I am referring to the sequential fractionation of a complex set of proteins, such as would be present in a tissue extract, by employing different physicochemical properties of proteins to affect each fractionation dimension. One of the most commonly utilized multidimensional fractionation techniques is 2D gel electrophoresis (2D-GE) where the proteins are first separated by isoelectric point and then by mass. While most often used as an analytical technique, 2D-GE can also be thought of as semi-preparative in that specific spots can be cut out of the gel and used as a source of material for protein identification. The first dimension, isoelectric focusing (IEF), is becoming quite popular for preparative fractionation schemes for the same reasons that it is used in 2D-GE: it is very reproducible, and the physicochemical property on which the fractionation is based is not influenced by the mass of the fractionated proteins, thereby freeing this property for downstream fractionation methods. Given the flexibility and utility of this technique, preparative IEF fractionation may be a valuable addition to your lab’s repertoire of protein separation methods. |
|
|
Focus on downstream applications |
|
|
| One of the first things to explore before embarking on a search for an appropriate IEF fractionator is exactly what applications downstream of IEF you intend to employ. This might sound obvious but knowing what you plan to do with the fractionated proteins will narrow your choices considerably. For example, if your intended application is the purification of active enzymes, you only need look at those instruments that are designed to run in the absence of denaturants. Likewise, if your intent is to pre-fractionate proteins prior to separating them further on narrow pH-range 2D gels, your best bet is the fractionator that prefers protein mixtures in 2D-GE-compatible buffers. Another helpful parameter to consider prior to shopping is your expected sample load, i.e. how much protein you envision running at a time. IEF fractionators can differ markedly in the quantity of protein they can handle, so if you plan to use the instrument as a first step in the purification of a rare protein from bovine brain, you don’t even need to look at nano-bore chromatofocusing instruments. |
|
|
Sizing up the equipment |
|
|
| Once you have nailed down your expected downstream applications and sample size, a reasonable next step is to find out what’s available. Literature and internet searches are excellent ways to obtain this information. In the broadest sense, there are 3 main categories of preparative IEF fractionators: 1) the Rotofor (BioRad, Hercules, CA)-type, characterized by large (up to 3 g) protein load capacity, large (18-55 ml) sample volume, fractionation of proteins in the native state, and considerable overlap with regard to the proteins in the fractions; 2) the ZOOM® IEF Fractionator (Invitrogen Carlsbad, CA)-type, based on a design reported by Zuo et al. and (1, 2) characterized by smaller (1-2 mg) protein loads, smaller (3 ml) volumes, fractionation of proteins in denaturing buffer, and more discrete fractions; 3) Chromatofocusing (CF) is a little more difficult to pigeon hole as CF is more of a method rather than a particular instrument. CF can be carried out with conventional HPLC or FPLC instruments with appropriate columns and buffers. It is a potentially very useful fractionation method in that it is extremely flexible and has high loading capacity (up to 100 mg protein/ml). CF can be carried out on anion exchange or cation exchange columns, and eluting buffers containing ampholytes or mixtures of other buffers can be individually tailored to maximize the fractionation of a given protein mixture. |
|
|
Figuring out the costs |
|
|
| For cost, expect to spend around $4,000 for the Rotofor-type and around $2,000 for the ZOOM®-type fractionators (The Nest Group, Inc. markets a similar device). The cost to get started with CF is more open-ended as it depends on what ancillary equipment you have on hand. If your lab already has an HPLC or FPLC, it may just be a matter of purchasing a column and some reagents. However, if you need the whole set-up, the price rapidly surpasses the $20,000 mark. In addition to purchase price, there is also the matter of cost per run. Although the Rotofor does not employ single-use parts per se, certain pieces of the instrument must be replaced after every 5 runs to ensure uninterrupted operation. The ZOOM® fractionator on the other hand does employ single-use parts, making each run cost around $50. For CF, it’s mainly a matter of reagent costs. No matter what the cost per run for any of these techniques, the good thing is that none of them can be considered high throughput, so operation costs don’t add up too quickly. |
|
|
Conclusion |
|
|
| In summary, preparative IEF can be an excellent addition to multidimensional fractionation schemes. The heightened interest in this technology has not gone unnoticed by the vendor community, resulting in a broadened selection of instruments from which to choose. While this is unquestionably a plus, it also makes the decision of which instrument or technology to go with a bit more challenging. However, deciding what your specific goals are and finding out the options available can make this decision much easier. |
|
Michael Campa, Ph.D.
Asst. Research Professor of Radiology
Duke University Medical Center
Related Product Links:
 2-D Conversion Kits
2D Electrophoresis Accessories
2-D Electrophoresis Cells
2-D Gel Analysis Software
2-D Gel Electrophoresis Systems
2-D Protein MW Markers
IEF Buffers
IEF Disks
IEF Loading Buffers
Isoelectric Focusing Gels
Isoelectric Focusing Systems
Preparative 2-D Electrophoresis Systems
SDS-Page Sample Prep Kits
More Articles:
Buying Tips Archives
Product Reviews
Feature Articles
|
