manufacturer :       BioRad

model :                   Rotofor Cell

annotation :           Dokumente engl.

The Rotofor cell is a unique preparative protein purification device that separates proteins in free solution by liquid-phase isoelectric focusing (IEF). Whether used alone or as a part of a broader purification scheme, the Rotofor offers rapid, simple, and effective fractionation and purification of even low-abundance proteins from complex protein mixtures.

Simplified Protein Purification and Sample Prefractionation

The Rotofor system is ideal for protein purification or prefractionation from crude or partially purified samples.

• Two interchangeable focusing chambers — mini and standard — process microgram to gram quantities of protein
• Easy setup and short run times enable 500-fold purification in <3 hours; a vacuum fraction collector instantly collects the resulting fractions
• The design of the Rotofor focusing chamber overcomes the diffusion, thermal convection, and precipitation problems that interfere with resolution in other preparative focusing techniques
• The patented ceramic cooling finger preserves biological activity, reduces diffusion and eliminates thermal convection
• The unique membrane core stabilizes proteins in 20 focused zones and facilitates free-solution collection without mixing
• Proteins may be purified in their native, soluble forms; otherwise, detergents or denaturants can be used to enhance protein solubility

The Rotofor system includes an 18 ml (mini) and 60 ml (standard) focusing chamber, harvesting box, and accessories.

Integrates Into Any Purification Scheme

The Rotofor rapidly isolates proteins of interest from the bulk proteins in complex biological samples and provides purified, biologically active proteins for functional and structural studies, antibody production, and sequence analysis.

• As an initial purification step, the Rotofor separates proteins of interest from bulk contaminants in crude samples; in such cases, optimal results are achieved when purification in the Rotofor is paired with analysis of the samples and fractions using Bio-Rad's precast IEF gels for preliminary pI determination
• Selected fractions from an initial run may be collected, pooled, and refractionated, resulting in up to 1,000-fold purification
• Rotofor fractions may be easily subjected to further purification by gel electrophoresis, chromatography, and other methods
• As a final purification step, the Rotofor eliminates specific contaminants that might be difficult to remove by other means

Effective Enrichment of Low-Abundance Proteins

The Rotofor separates complex protein mixtures into 20 distinct fractions by liquid-phase IEF. Because the Rotofor system can process up to gram quantities of protein and effectively concentrates proteins into discrete zones based on their pI, the Rotofor is a proven and effective method for the enrichment of even low-abundance proteins.

Interchangeable Focusing Chambers

The central component of the Rotofor cell is the cylindrical focusing chamber, available in two sizes. The standard Rotofor chamber holds from 35 to 58 ml of sample, and is commonly used to fractionate samples containing a few milligrams to 4 g of total protein. The mini Rotofor chamber holds 18 ml of sample and is ideal for small samples containing micrograms to milligrams of sample. The mini chamber is also ideal for refractionation of samples harvested from an initial Rotofor run in the large chamber. Rotofor adaptor kits are available to convert the standard-size Rotofor cell to a mini Rotofor cell, and to convert the mini Rotofor cell to a standard-size Rotofor cell.

The Principle

Proteins are amphoteric molecules, meaning they carry positive, negative, or neutral charges depending on the pH of their local environment. For every protein, there is a specific pH at which the net charge it carries is zero. This isoelectric pH value, termed the pI, is a physicochemical characteristic of every protein. It is the amphoteric nature of proteins and their characteristic pIs that allow their separation by IEF.

Using Bio-Lyte ampholytes in an electrical field, the Rotofor system generates linear pH gradients to fractionate complex mixtures of proteins. For example, a protein with net positive charge in a particular region of the pH gradient will migrate away from the anode (+) and toward the cathode (–), giving up hydrogen ions (H ⁺ ) as it goes. When the net charge on a protein becomes zero, it ceases to migrate. If a protein should acquire a charge by diffusing in the pH gradient, the electrical field will drive it back to the pH that matches its pI. In this way, each protein in a sample becomes focused at its pI. Purified proteins are then harvested in free solution within specific pH intervals.

All Rotofor cells are shipped with a starter kit to familiarize new users with the cell. The starter kit contains a mixture of naturally colored proteins, a 10 ml bottle of Bio-Lyte ampholytes (pH range 3/10), a 1 ml protein sample, a 60 ml syringe, and a sample-loading needle. Use of the sample kit is demonstrated in the figure below.

A mixture of naturally colored proteins, including phycocyanin (blue isoforms, pI 4.5–5.0), hemoglobin (red isoforms, pI 7.2 and 7.6), and cytochrome c (orange isoforms, pI 9.5–10.0), was combined with Bio-Lyte ampholytes, pH range 3–10. The sample was injected into the standard (60 ml) focusing chamber (A). The proteins were focused (B) and after 1 hour of focusing (C), the three proteins had been separated. Using the Rotofor system's vacuum-assisted harvesting system, fractions were simultaneously aspirated into 20 individual test tubes (D).

Required Auxiliary Equipment

Accessory equipment required to run the Rotofor cell includes a 5,000 V power supply, such as the PowerPac HV power supply, a recirculating water chiller, and house vacuum.

Detailed Protocols for Purification

Numerous Rotofor purification schemes for recombinant and native proteins have been developed and published: see the examples accessible through the links at the top of the page. In addition, the Rotofor folder (bulletin 1555A) contains lists of references, a brochure, and sample application notes describing the use of the Rotofor in a variety of research projects.

Specifications