Frequently asked questions:
1. Is the sample diluted during the FFE process?
The dilution of the sample depends on the method used for FFE. Generally we get dilution factors between 1:2 and 1:4 in the IEF mode.
2. What is the recovery rate?
The FFE system is a closed process, that means all the sample applied can be collected again after the free flow electrophoretical separation. If you need higher protein concentration than your collected sample, we recommend Vivaspin™ ultrafiltration for the concentration of protein samples.
Here we get recovery rates of >98%.
3. Is the FFE system compatible to other techniques?
The system can be used prior all major techiques like HPLC, LC-MS, mass spectrometry (ESI / MALDI, depending on the protocol used), Electrophoresis (IEF / SDS PAGE, 2D-PAGE).
LC-MS for example can be fed directly after FFE separation.
4. How should the sample be prepared for FFE separation?
The chemical and physical properties of sample and separation media should be as similar as possible: Density, Viscosity and Conductivity should be equal, that means:
Just dissolve your sample in separation media or
Dilute your sample with separation media!
5. What is the maximum salt concentration?
The total salt concentration of the sample should not be more than 25mM for "standard" runs, but we can provide also special protocols for samples containing up to 1M and even 4M salt.
6. What if I have turbid samples?
Samples that contain cells, organelles or membranes are turbid by nature and don't have to be cleared.
Turbid protein samples indicate precipitation and/or insoluble components. Clearance by filtration or centrifugation is necessary.
7. Which additives are tolerable with FFE?
You can use almost any common additives and detergents like:
Urea up to 8M
0.1-1% of detergents like:
CHAPS, CHAPSO, Digitonin, Dodecyl-ß-D-maltoside,
Octyl-ß-D-glucoside, Triton-X-114 (IEF)
Up to 50 mM DTT
8. What is the upper size range of protein complexes/nano-particles/organelles that your system can purify?
The various techniques of FFE can be used even for the separation cells. There is no limitation of M.W. of protein complexes/nano-particles/organelles, to be separated with FFE!
9. Does FEE give more homogeneous complexes than HPLC, native gels?
FFE gives the chance for the isolation of “charge-isomers” of protein complexes, while preserving the biological activity.