January 15, 2020

Lab scale filtration for chromatography and ultrafiltration

By Cytiva

Reliable sample preparation and fractionation processes are key to the success of proteomic research. Learn how our lab-scale filtration solutions – for ultrafiltration, diafiltration, membrane chromatography, and protein fractionation – bring speed, efficiency, and cost savings to your life science research.

Cost-effective and innovative filter plates for chromatographic purification

Many tools and techniques are available today for protein purification. Development of a purification process requires an analysis of various strategies to isolate a protein of interest. Protein purification from a complex sample, such as cell culture or serum, requires more than one chromatographic step. Typically, the first step is ion exchange (IEX) or mixed-mode chromatography to fractionate the sample and yield a subpopulation of proteins containing the target molecule. This is followed by subsequent chromatographic steps to further fractionate the sub-population until a desired purity level is reached. These steps can include affinity purification and size exclusion chromatography, as well as IEX or mixed-mode chromatography to isolate the target protein.

Each step in the purification process requires optimization in order to maximize yield and purity of the target protein. Thus, small-scale purification experiments are used during the development and optimization phase to preserve precious sample while providing crucial information. Use AcroPrep™ Advance 96-well filter plates combined with chromatography membranes as an efficient tool for fractionation of small-volume protein samples. The format can be used to develop protein purification strategies and/or as a platform for moderate- to high-throughput needs.

AcroPrep™ Advance 96-well filter plates (Pall™ Life Sciences products) offer excellent consistency and reproducibility. Other features include:

  • Well design enhances well-to-well consistency for sample processing and recovery.
  • Outlet tips minimize the formation of hanging drops, reducing the chance of cross-contamination after filtration.
  • Optimized membrane configurations for assays, such as multiplexing.
  • Individually sealed membranes and biologically inert housing minimize the possibility of sample cross-contamination or loss.
  • Designed to meet SBS/ANSI guidelines for compatibility with standard processing equipment.
  • Smooth top surface, textured window for labeling, and A1 corner notch allow for easy identification.

Laboratory ultrafiltration membranes

Biomolecule purification involves a complex series of steps where targets are selectively separated through sequential processes. The process by which separation is performed often creates a need for the sample to be desalted or concentrated to prepare the biomolecule sample for the next step in the purification process. We offer several technologies for efficient sample concentration, desalting, and buffer exchange, including ultrafiltration (UF) spin filters and UF multi-well filtration plates.

Ultrafiltration is a membrane separation technique that involves forcing a protein solution, usually by centrifugation, through a membrane with a defined molecular weight cutoff. Selection is by molecular size, although other factors, such as molecule shape and charge, can also play a role. Molecules larger than the membrane pores in the UF membrane will be retained at the surface of the membrane while solvent and smaller solute molecules will freely pass. This molecular exclusion at the UF membrane surface leads to concentration of the protein solute in the retained fraction (termed the retentate) and can be recovered from above the membrane. In other words, the protein does not pass through the membrane, whereas the other buffer components do. This method is usually relatively rapid and does not adversely affect the protein sample. The filtrate can also be recovered and evaluated.

One of the other common applications for these devices is buffer exchange or desalting. Salt-sensitive downstream applications such as gel electrophoresis, nuclear magnetic resonance (NMR), X-ray crystallography, and mass spectrometry require desalting of samples. One simple way to achieve this is to use a centrifugal device, such as our Nanosep™ Centrifugal Devices (Pall™ Life Sciences products).

Our ultrafiltration centrifugal devices facilitate pure product with > 90% recoveries in just minutes.

Compared to non-membrane processes (resin-based chromatography, dialysis, solvent extraction, or centrifugation), ultrafiltration:

  • Is far gentler to the molecules being processed
  • Does not require an organic extraction, which may denature labile proteins; maintains the ionic and pH milieu
  • Is fast and relatively inexpensive
  • Can be performed at low temperatures (for example, in the cold room)
  • Is very efficient and can simultaneously concentrate and purify molecules

We offer a complete range of ultrafiltration devices suitable for various sample volumes. These centrifugal devices are specifically engineered to provide faster flow rates and easier handling. Devices are available with low protein binding Omega™ ultrafiltration membrane.

Small-scale chromatographic protein fractionation

Fractionation using ultrafiltration of complex protein solutions and separation of unbound or unincorporated label from protein samples are some of the main applications of these centrifugal devices. It is important to note, that this method is also valid to remove cellular debris after cell lysis to partially purify protein product found in the filtrate. This is important because if the two proteins that need to be fractionated from one another were close in size, then it would be impossible to purify the two from one another. More expensive chromatographical methods would have to be used in that instance.

See the first section of this article for purification strategies of proteins of similar size. Typically, products are now ready for further processing or direct use in downstream applications, such as Western blotting or ELISA.

Discover the benefits of the AcroPrep™ Advance 96-Well Filter Plates for Protein Purification

Diafiltration for desalting and buffer exchange

Diafiltration is a technique that uses ultrafiltration membranes to remove, replace, or lower the concentration of salts or solvents from solutions containing proteins, peptides, nucleic acids, and other biomolecules. The process selectively utilizes permeable (porous) membrane filters to separate the components of solutions and suspensions based on their molecular size. An ultrafiltration membrane retains molecules that are larger than the pores of the membrane while smaller molecules such as salts, solvents and water, which are 100% permeable, freely pass through the membrane.

Conventional techniques used for salt removal or buffer exchange such as membrane dialysis and column-based gel filtration can be effective but have limitations. With diafiltration, salt or solvent removal as well as buffer exchange can be performed quickly and conveniently. Another big advantage of diafiltration is that the sample is concentrated on the same system, minimizing the risk of sample loss or contamination. There are several ways to perform diafiltration. While the end result may be the same, the time and volume required to complete the process may vary considerably. It is important to understand the differences in the methods used and when to choose one over the other.

Learn more in our brochure.