Ultrafiltration centrifugal filter units are widely used for the concentration, purification, and desalting of proteins, DNA, and other molecules in laboratory research. In this article, we will discuss what an centrifugal filter is and how to use these devices effectively.

What is ultrafiltration?

Before we dive into what is an centrifugal filter. It is essential to understand what ultrafiltration is and how it differs from microfiltration.

Microfiltration

Microfiltration (MF) involves the utilization of microporous membrane media to eliminate particles or organisms with sizes ranging from 0.1 μm and 10.0 μm from a solution. The pore size of these membranes is bigger than that of ultrafiltration membranes (0.01 μm to 0.1 μm).

Microfiltration membranes find widespread application in prefiltration and sterile filtration processes within the biopharmaceutical manufacturing industry. In cell experiments, microfiltration is most commonly used for sample and culture medium sterilization (0.1 μm microfiltration can remove mycoplasma).

Additionally, microfiltration is employed to efficiently remove intact cells and debris from cell lysates prior to nucleic acid and protein analysis. This step is crucial in ensuring accurate and reliable results during downstream applications. By employing microfiltration, researchers can obtain purified samples, enhancing the sensitivity and specificity of their nucleic acid and protein studies.

Ultrafiltration

Ultrafiltration (UF) is a variety of membrane filtration in which forces such as pressure or concentration gradients lead to a separation through a semipermeable membrane. The ultrafiltration membrane is capable of excluding molecules with sizes ranging from 1-1000kDa (MW), and allowing small molecules like salt and water to pass through. It has been widely used in processing macromolecular samples, especially protein solutions.

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When compared to the precipitation method, the ultrafiltration method is milder, avoiding cause of phase changes that could potentially result in the inactivation of biomacromolecules. Moreover, ultrafiltration can complete the desalination of the solute while concentrating.

The application of ultrafiltration is not limited to the concentration and purification of protein samples, but is also commonly used in the preparation of nucleic acid samples, including molecular cloning and plasmid purification.

What is centrifugal filters?

An ultrafiltration centrifugal filter is a device that utilizes centrifugal force to gently drive small to medium volume solutions through ultrafiltration membranes for rapid concentration, diafiltration and buffer exchange. It consists of a lid, a filter device and a centrifuge tube.

Centrifugal filter structure diagram

Centrifugal filters can be employed for both permeate samples or retained samples.

When the target molecule is significantly smaller than the pore size of the membrane, centrifugal filters can be used for heat removal, clarification and separation of macromolecular pollutants, the sample permeates through the filter membrane and retained in the centrifuge tube.

Conversely, if the target molecule is much larger than the membrane pore size, small molecule pollutants will be filtered out and the target molecule will be concentrated in the filter device.

How to choose an centrifugal filter unit?

Starting Volume

Choose different centrifugal filter sizes based on the volume of your sample. Using fix-angle rotor centrifuge or swing-bucket rotor centrifuge will follow different protocols, please refer to the diagram below to determine the maximum starting volume:

Diagram 1: Maximum Starting Volume

MWCO

Although there are many factors affect interception and product recovery, we generally recommend choosing an ultrafiltration membrane with a molecular weight cut-off (MWCO) that is 2-3 times smaller than the molecular weight of the target macromolecule that want to be concentrated. This choice ensures better results and the efficiency of the ultrafiltration process.

 Diagram 2: MWCO and Target MW 

Want to know more details about how to choose centrifugal filters and how factors like sample concentration, transmembrane pressure, molecule composition and more affect the retention capability? Read this article: How to choose MWCO filters?

How to use centrifugal filter units?

Once you have selected the appropriate centrifugal filter unit based on MWCO, volume of your sample etc. it's time to proceed with its usage. Typically, ultrafiltration centrifugal filters come with a small amount of glycerol for protection and storage, and it is crucial to rinse the device before use to eliminate any glycerol residue.

Step 1: Preparation-Rinse Before Use

1. Add 4mL pure water, deionized water or buffer solution to the ultrafiltration device.
2. Centrifuge at 4000xg (swing bucket) or 5000xg (fixed angle 45°) for 5-10 minutes.
3. Discard all liquids from the ultrafiltration unit and centrifuge tubes.
4. If any interference persists, wash with 0.1N NaOH under the same conditions as mentioned above, followed by rinsing 2-3 times with ultra-pure water, deionized water, or buffer.
5. Ensure that the ultrafiltration membrane remains wet once wetted until it is entirely used.

Step 2: Add Sample

Carefully add your sample into the filter device, taking care not to overfill it (Fix-angle rotor centrifuge should add less volume as shown in Diagram 2 above). After adding the sample, securely seal the filter's lid to prevent any leaks during centrifugation.

Step 3: Centrifugation

After loading your sample into the filter device, you should determine the appropriate centrifuge speed and duration based on the guidelines and specific properties of your sample.

Here is the recommended G-force for reference:

 Diagram 3: Recommended G-force

During centrifugation, it is crucial to maintain balance among your samples. To achieve this, place an equal number and equal mass of samples on opposite sides of the centrifuge rotor. This balancing act ensures the rotor remains stable, preventing any potential damage to your samples or the centrifuge itself.

Step 4: Retrieve the Sample

Once the centrifugation process is finished, retrieve your samples from the filter. Avoid opening the lid while the rotor is still in motion, wait until the centrifuge rotor has slowed down or come to a complete stop.

Carefully remove the filter from the centrifuge and gently open its lid. Utilize a pipette or an appropriate tool to extract the sample from the filter device or the centrifuge tube, depending on your requirements. When pipetting from the filter device, take special care NOT to let the pipette tip touch or disrupt the membrane. When extracting the sample, pipette only from the edge of the filter.

Exercise utmost caution while handling your samples to prevent any potential contamination or loss. Finally, store your samples properly, following the recommended storage conditions to maintain their integrity and quality for future use.

Problem Solving

Protein Precipitation

If the protein is concentrated too rapidly or to a high concentrated, it may lead to protein precipitation. To prevent this, it is recommended that the final protein concentration after concentration should not exceed 20mg/ml. For proteins that are sensitive to concentration speed and prone to precipitation, the following improvements are suggested:

1. Reduce the centrifugal force to 30%-50% of the recommended centrifugal force.
2. Consider using an ultrafiltration filter with a larger Molecular Weight Cut-Off (MWCO). For example, if you initially selected a 3k MWCO, you can switch to a 10k MWCO.
3. During the concentration process, take out the ultrafiltration filter, gently mix the sample with a pipette several times to prevent blocking the filter membrane, and then continue with the centrifugation. Repeat this mixing process if necessary.

Loss of Protein

The minimum recommended starting protein concentration is 25ug/ml. Please ensure that the initial concentration of your sample is greater than this value. Retain the liquid in the centrifuge tube for further analysis.

If the target protein is in the centrifuge tube, please check the following:

1. Have you chosen an ultrafiltration filter with an appropriate MWCO? It should be 2-3 times smaller than the molecular weight of the target macromolecule.
2. Are you using the correct G-force during Centrifugation?
3. Is this the first time you are filtering the protein? Sometimes, the protein's conformation can affect the concentration efficiency, and in such cases, it is recommended to use an ultrafiltration filter with a smaller MWCO.

If the target protein is not found in the centrifuge tube, please check the following:

1. Is the initial protein sample concentration greater than 25ug/mL?
2. Has the target protein precipitated? If yes, please refer to the above explanation about protein precipitation for specific solutions.

Can centrifugal filters be reused?

We do not recommend reusing centrifugal filter units. Attempting to reuse them can result in decreased filtration efficiency and may potentially lead to sample contamination.

However, we are aware that some users have reused centrifugal filter units for the same sample up to a maximum of 3 times, only for reference. For optimal results and to ensure the integrity of your samples, it is best to use centrifugal filters only once.

Conclusion

In laboratory research, ultrafiltration centrifugal filters find extensive application in concentrating, purifying, and desalting various biomolecules. This article discusses the differences between ultrafiltration and microfiltration, and provides guidance on how to select and use centrifugal filters, which can serve as a reference for researchers and professionals. Cobetter develops and manufactures Briscale® centrifugal filters, which have reliable performance and high protein recovery, making them an excellent choice for ultrafiltration needs.