Free Flow Electrophoresis System

FFE is an acronym for Free Flow Electrophoresis.

• of simple or complex samples of any charged or chargeable molecules/particles (e.g., organelles, proteins, protein complexes, membrane proteins, peptides, etc.)
• in a thin film of aqueous liquids in the absence of any kinds of stationary phases like gels
• under both native as well as denaturing conditions
  
  

FFE can be used to separate all kinds of charged samples, especially proteins, peptides, protein complexes, membranes and organelles.

The BD™ Free Flow Electrophoresis System can be used for different modes of operation which are supported by validated protocols. The mode the separation is based on different physicochemical properties (please refer to the following table):

1) FFE is a liquid based sample separation/preparation technique, i.e. it is compatible with all kinds of downstream separation technologies like 2-DE or LC-MS.

2) The separations are very fast.

3) Liquid phase separations enable very high recoveries of sample components.

“Traditional” separation techniques like chromatography or gel electrophoresis work in a batchwise mode, i.e. an injection or loading-step is followed by a separation-step, the so-called run. FFE is typically used in a continuous mode, i.e. the injection-step as well as the separation-step occur continuously and simultaneously. In this context, the expression “run” is not really appropriate, because as soon as a steady state is reached the fractionated sample can be collected whenever you want as long as you want.

The reasonable concentration range is approximately 1mg/ml- 5 mg/mL for standard protocols. We recommend starting with a concentration of approximately 1-2 mg/mL. Special protocols are available for low sample amounts down to 50µg/ml.

It is recommended to start with approximately 5 min for standard protocols. Defining an upper limit is difficult for a continuous process. For this, we recommend starting with a sample application time of at least 30 min-1 hour.

Between 1-10 mL/hour should be fine.

Theoretically, it is around 80-100µL assuming a typical application speed of 1 mL/hour, and a minimum application time of 5 min. However, the dilution of the separated species inside the fractions will be higher and the recovery lower than with mL amounts of starting sample. Even lower sample volumes may be possible if special separation protocols are applied.

It is approximately 8 µg assuming a typical application speed of 1 mL/hour, a minimum application time of 5 min, and a minimum sample concentration of 100 µg/mL.

Theoretically, based on the values mentioned above it should be between 0.1-10 mg/h.

Assuming that you will “inject” 100µL of sample within approximately 5 minutes, you will probably have to collect for 10 minutes to get a reasonable recovery. The flow rate of the separation buffers is typically around 60-100 mL/hour. (i.e. approximately 1mL per fraction per hour; i.e. 170µL per fraction per 10 minutes). Lower collection volumes may be possible if special separation protocols are applied.

Maximum collection volume is limited only by the amount of sample you have and not the fraction collector. The unit can operate in a continuous flow and continue collecting as long as you wish to over time.

Collection is in a standard 96 well format. The unit accommodates standard depth or any deep well format.

We recommend the exclusive use of BD proprietary FFE reagents that are designed specifically for use with the BD FFE system. Our proprietary reagents consist of low Mw organic acids and bases. These molecules are extremely well characterized (MW, pI, purity, toxicity) and are compatible with standard mass spectrometer. Commercially available ampholytes, on the other hand, are uncharacterized mixtures of poorly-characterized polymers. In addition, our proprietary reagents have a significantly higher buffering capacity than standard ampholyte mixtures. This allows FFE separations to be performed with higher salt concentrations that do not interfere with the separation.

It is possible to use ultrafiltration or reverse phase material to remove our proprietary reagent additives or detergents. Clean up strategy will depend highly on the objective of the clean up; either reduction or complete removal of additives. For many downstream applications, clean up either is not required or for some processes, typical steps (e.g. zip tips before MS) also remove the FFE buffer components.

Yes. The instrument can work in native mode as well as in denaturing mode using a variety of detergents to suit different solubility needs.

All commercially available non-ionic reagents which are used for standard applications are compatible with the BD FFE System. Some examples are Triton X-114, NP-40, ASB-14, Zwittergent® 3-10, CHAPS, etc. Their concentrations have to be optimized for each application. A concentration of 0.1% is  a good starting point.

For standard protocols, it is less than or equal to 0.1 pI, for special protocols, it is less than or equal to  0.03 pI.

How many fractions do I finally get running the BD™ Free Flow Electrophoresis System?

The unit flows continuously and collects all fractions in a 96 MTP format. To guarantee stable and reproducible separations, however, the number of sample-containing fractions is limited to approximately 70.

Yes, it is possible to reduce the “net” width of the separation area, i.e. the number of sample-containing fractions to anywhere from 13 to 70 using appropriate media.

Individual fractionated proteins are typically limited to 1-2 wells, but this depends on the intermolecular interaction-tendency of the proteins as well as the titration curve profile of the proteins, i.e. the migration speed of proteins around their pI.

Yes. The BD™ Free Flow Electrophoresis System is simply a high-end separation and enrichment technique. Fractions can then be cleaned (i.e. detergents, salts, etc.) and prepared for further analytical techniques.

Yes. The BD™ Free Flow Electrophoresis System is simply a high-end separation and enrichment technique. Fractions can then be cleaned (i.e. detergents, salts, etc.) and prepared 2D-LC/MS/MS thus enabling enhanced resolution of complex mixtures by multi-dimensional (MudPIT) chromatography techniques.  For LC-MS the samples can be fed directly into the instrument after FFE to the system.

Talking about "run“ time doesn’t make sense for FFE, because FFE is a continuous technique which means that a “run” can be as long as you want. Talking about residence time (i.e. the time that a sample spends in the separation chamber) is better suited. For the standard protocols it is approximately 15-20 min. This means that the total time between sample application and sample fraction collection is approximately 20-30 min. The exact time can be calculated easily based on the volume of the separation chamber and the flow of the separation media. In addition, internal “marks” with dyes like SPADNS and/or with colored pI markers can be used to visualize the separation process. Lower residence times down to 5 min may be possible if special separation protocols are applied.

2000 Volts.

The standard protocols are designed for FFE-IEF (pH 3-10) of protein samples in both denaturing and native conditions. However, the unit is capable of running in ZE (Zone Electrophoresis), or ITP (Isotachophoresis) modes, too.

The chamber is 0.1 or 0.5mm thick, 100mm wide and 500mm long. However, the “net” width of the separation area can be varied from 13-70mm.

Yes, but to a tolerable degree. Please see next question for details.

Assuming a sample application speed of 1mL/hr and a media flow rate of 100mL/hr (~1mL/hr/well for 96 well plate) and typical distribution of a protein within your sample to 2-3 fractions, you would end up with a 2-3 fold dilution for each protein. This is independent of the number of proteins in your sample. Please remember that 100% of each specific protein was in 1mL of solution and ends up in 2-3mL of solution. Hence, this is a 2 to 3-fold dilution.

The starting amount of sample does not have anything to do with dilution as long as the sample application time (e.g. sample volume) is high/fast enough. However, recovery is also limited by adsorption phenomena. To achieve reasonable recovery, your total sample collection time should be a bit (5-10 minutes) longer than your total sample application time. The reason is that proteins in the middle of the chamber need a different time than proteins that flow close to the chamber walls. This translates to some additional dilution.

Both the internal chamber of the FFE and the fractionation outlets have a relatively low surface area compared to other techniques. In addition, adsorption of material is reduced by using appropriate surface materials and additives. Thus, the recovery of the sample will be nearly 100% for most of the samples if the “run” time and sample amount are not at the lower limit.

Each and every protein behaves differently regarding surface adsorption. This is the only reason for loss of protein on our system. Thus, loss due to adsorption is unpredictable. However, this is addressed as best as possible by treating the internal chamber surfaces and including an additive to the separation media which reduces surface adsorption. These treatments do not affect the functional state of proteins under native conditions either. In addition, our fraction collection tubing is Teflon coated, which further minimizes protein adsorption.

Precipitation is significantly low compared to other technologies since all separations are done in liquid phase using appropriate separation media and not relying on solid matrices like membranes or gels for separation. If precipitation occurs this will be clearly visible through the transparent front plate of the separation chamber and the separation conditions can easily be optimized to suppress the effect.

No, if you follow the protocols.  If the media flow rate or the chamber temperature is too low, it may happen from time to time.

Typical buffers can in some cases include glycerol, HPMC and / or (thio)urea, in addition to our proprietary reagents according to the standard protocols. However, a large number of protocols are compatible with the main downstream analytical techniques or refer to easily removable components.

All buffers with a total salt concentration of 30 mM are compatible with the BD™ Free Flow Electrophoresis System.

The sample can be mixed with the red dye "SPADNS".  As soon as you observe red drops at the fractionation point, you can start collecting your sample! BD offers this dye as part of the control kit.

Is it possible to use markers to know when to stop collecting fractions? Should we just calculate how long we need to collect for to get reasonable recovery?

See previous question.

We provide a set of colored pI-markers that can be used to control the quality of the FFE separations.  We also provide validated protocols containing the appropriate parameters (e.g.  flow rate, voltage, etc.) and a characteristic pI quality test for the instrument.

The average set up time including cleaning and quality control test prior sample injection is approximately 1-2 hours. The cleaning/shut down procedure after using the FFE takes up to 30 minutes.

The whole system is washed at the end of the day/ of an experiment by actively pumping pure separation media and pure water through the system for 20 min. Subsequently, pure water is used to passively rinse the system overnight.

The system is cleaned by opening the separation chamber and washing it manually using water, isopropanol, and petrol ether. This takes approximately 10-15 minutes.

Yes. The BD™ Free Flow Electrophoresis System is simply a high-end separation and enrichment technique. Fractions can then be cleaned (i.e. from detergents, salts, etc.) and prepared for further analytical techniques (e.g.  2D-LC/MS/MS) thus enabling enhanced resolution of complex mixtures by multi-dimensional (MudPIT) chromatography techniques. For LC-MS the samples can be fed directly after FFE to the system.

Zone electrophoresis is a FFE separation mode which enables to separate charged particle in constant pH. The net charge densities of the particles are used to separate them from each other.

No. But please note that generally for obtaining best results for organelle separation we need gentle and native sample preparation. A large number of protocols for organelle sample preparations are available and can be obtained by literature research. References can be found in our organelle publication list.

Due to the fact that freezing and thawing damages the membranes of the organelles, we do not recommend using of frozen sample for organelle separation.

Each charged or chargeable particle can be separated by FFE.

Depending on the amount of sample that has to be separated it can take from 5 minutes for analytical application up to hours for preparative applications.

Because of specific separation of sample components the proteolytic enzymes can be separated easily from other subcellular components. This avoids the early sample degradation.

There are no sample source limitations for organelle separation using FFE.

We recommend 2 mg/ml protein concentration. Typical protein concentrations are in the range of 1-3 mg/ml.

According to an experience we need at least 1g freshly excised tissue. For separating organelles from cultured cells we need at least 1xT₁₇₅ flask (1-5x10⁷ cells).

Using FFE for organelle separation allows application of every analytic method, e.g. electron microscopy or enzyme assays.

Yes, because of the native conditions during the organelle separation by FFE the biological activities of the enzymes in organelles are preserved and can be determined by using enzyme activity assays.

No, since the whole tissue homogenate contains large charged sub components and we recommend to pre-purify the sample by differential centrifugation prior to separation using in FFE-Zone Electrophoresis.

Each charged or chargeable particle can be separated by FFE.

The post-FFE fractions can be sedimented by centrifugation and gently resuspended in any desired buffer.

The post-FFE fractions can be sedimented by centrifugation and gently resuspended in any desired buffer amount to get desired sample concentration.

A huge variety of protocols are already published and by contacting our customer service every FFE-user is able to get the appropriate protocol for his/her application.

You can find more detailed information about FFE and its application at: www.bd.com/proteomics.

Following an appropriate sample preparation, each specimen can be separated by FFE.

Using the net charge density of the samples enables FFE to separate particles with same size shape and densities from each other.

FFE fractions are collected in standard 96 well format plates. Depending on the separated sample volume it can be collected in 200 µl up to 4 ml-well plates.

Yes, up to certain extent but the dilution effect can be dismissed because you can easily sediment and resuspend the post-FFE fractions in a desired buffer volume.

A protein isoform is defined as a variant of a single polypeptide which generally alters its function to increase the diversity of functions and regulate the activities of proteins that may be caused by a disease.

More than 90% of naturally occurring isoforms arise from post-translational modifications (PTMs) and less than 10% from mRNA splice variation. Most common PTMs include phosphorylations and glycosylations; but several others such as acetylations play an important role as well.

The sample may be modified during sample handling. Oxidation and deamidation of the sample are common modifications and are not a FFE-specific phenomenon. In many cases, recombinant production of proteins results in several protein isoforms (modification at the N-terminus, deamidations, etc.).

Accurate protein identification sometimes requires careful discrimination between closely related protein isoforms that differ by as little as a single amino acid substitution or post-translational modification. Additionally, a critical request is the separation of isoforms as intact and functional proteins.

FFE uses the isoelectric point, net charge and electrophoretic mobility of protein isoforms for separation in an electric field.

Running an electrophoretic titration curve and/or IEF-PAGE and/or 2D-PAGE provides information about isoelectric point and electrophoretic mobility of the sample. This information is essential for choosing the appropriate FFE separation mode (Zone Electrophoresis, IEF, etc.) and sample injection (cathodic, anodic or central injection).

Yes, A and B forms of Beta-Lactoglobulin are applicable with high reproducibility.

We offer different protocols with a broad range of pH gradients for separation of protein isoforms with the BD™ FFE System.

Protein isoforms with different isoelectric points can be separated using isoelectric focusing mode (IEF). The separation resolution of BD™ FFE-IEF goes down to 0.02 pH units. Protein isoforms with similar isoelectric points but different electrophoretic mobility can be separated in the non focusing FFE-mode of zone electrophoresis.

Depending on protein abundance, the minimum amount of the sample which can be injected into the BD™ FFE System is between 30-500 µg per hour.

Yes, because separation of protein isoforms can be performed under native conditions. All buffer components are compatible with downstream processes such as ELISA or any other kind of assays.

Yes, because native conditions are maintained with FFE, the biological activities of the sample are preserved and can be determined by down stream application.

After separation of protein isoforms by FFE, the buffer of FFE fractions can be exchanged using the current protocols.

A large number of protocols are already published and by contacting us at BDFFE@europe.bd.com every BD FFE-user will be provided with appropriate protocols for their application.

A large number of FFE applications are already published. A list of related publication can be found at http://www.bd.com/proteomics/references.

FFE fractions are collected in standard 96 well format plates. Depending on the separated sample volume it can be collected in 200 µl up to 4 ml-well plates.

For most techniques of downstream analysis FFE fractions can be used without any concentration of the sample. If needed FFE fractions can be concentrated using current protocols or commercially available materials.

Any charged or chargeable particle can be separated by the BD™ FFE System.

Membrane proteins are associated with cellular membranes. They are the interface between cells and their surrounding environment. Four main classes exist: integral, anchored, associated, and transitory membrane proteins. The biochemical analysis of (integral) membrane proteins is hampered because of the high hydrophobicity of membrane proteins. Two main tasks have to be performed: (i) the extraction of membrane proteins from their hydrophobic lipid environment and their solubilization in the aqueous environment; (ii) the membrane proteins have to be kept in solution and prevented from aggregating throughout the whole separation procedure. These challenges increase with increasing hydrophobicity of the analyzed proteins. Thus, the analysis and separation of integral membrane proteins, especially those containing several α-helical transmembrane domains, is very complex.

Membrane proteins have the tendency to precipitate at their isoelectric point. Therefore a novel separation mode on the the BD™ FFE System called, interval zone FFE was developed. In contrast to the isoelectric focusing (IEF) mode, this separation is carried out at a constant pH relying on the net protein charges. The applied pH of the separation buffers is selected in such a way that it is different from the isoelectric point of the proteins to be separated thereby maintaining proteins in solution which otherwise would precipitate. For further information on specific protocols, contact us at BDFFE@europe.bd.com.

BD™ FFE System is a technology which does not have any interaction with a solid separation matrix, which is the case for many other separation techniques. Matrix-free separations eliminate non-specific absorption and is a major feature of the BD™ FFE System. Constant sample application keeps the proteins in solution and the low sample absorbance provides access to proteins with extreme physical-chemical properties (like membrane proteins).

The total salt concentration should not exceed 30 mM. It is recommended to use non-ionic or zwitter-ionic detergents.

FFE separation of multi-protein complexes under native conditions is possible and published. For further information on publicatins go to www.bd.com/proteomics/references.

With Interval Zone Electrophoresis separation time is approximately 10 minutes.

Typical sample range is between 0.3 - 0.5 mg protein per run.

Yes, after fractionation the sample is maintained in solution. The media used for separation are compatible with subsequent proteolytic digestion. Furthermore the detergent-free separation media facilitates the coupling with liquid chromatography and tandem mass spectrometry since no detergent removal is required.

In the case of denaturing separation conditions urea/thiourea can be easily removed with SPE (solid phase extraction).

A large variety of protocols are published. By contacting us at BDFFE@europe.bd.com every BD FFE user will have access to appropriate protocols for their application.

You can find more detailed information about FFE and its application at: www.bd.com/proteomics/references