Modified
Dec 2012
RW

Advances in Free Flow Electrophorsis since 35 years

 Highly Versatile Solutions for your Separation Needs

Effective protein research demands high throughput, reproducibility and outstanding sensitivity.

The FFE system offers a unique combination of performance and revolutionary technology to transform the quality,
speed and ease of execution of all your laboratory’s proteomic procedures, scientific data handling and analysis.

1. Highest resolution for protein isoform separation

2. Separation of Isoforms (new protocol)

3. Very high throughput for denaturing separations

4. New protocols for protein complex separations

5. Customized ultra flat pH gradients

6. Reproducibility and Recovery

1. Highest resolution for protein isoform separation

1.a) Native separation of Protein Isoforms at pH extremes

Alkaline Ampholyte pH gradient for native IEF separation of Elastase Isoforms (pI 10.4); FFE conditions
2000V /19 mA, transit time 5 Minutes; chamber 500mm x 50mm x 0.15mm

native IEF separation of Elastase Isoforms (pI 10.4) , visualized on a Serva IEF gel
first dimension FFE-IEF pH 10.02 - 10.77, second dimension PAGIEF pH 3-11

Flat Ampholyte pH gradient for native IEF separation of Ovalbumin isoforms; FFE conditions
2500V /15 mA, transit time 7.5 Minutes; chamber 500mm x 50mm x 0.2mm

native IEF separation of Ovalbumine Isoforms (pI 4.5) , visualized on a Serva IEF gel
first dimension FFE-IEF pH 4.61 - 4.92, seciond dimension PAGIEF pH 3-11

1.b) Customized IEF separations for protein isoforms

Flat Ampholyte pH gradient of pH 4-6 for Isoform separations; FFE conditions:  2500V /12mA, transit time 6.5 Minutes;  chamber 500mm x 50mm x 0.175mm

Flat Ampholine pH gradient of pH 4-6 in the first dimension, second dimension PAGIEF 3-10

3. Separation of Isoforms (new protocol)

example 1:
 isoform separation of ß-Lactoglobulin,
isoforms at 5,15 (fraction 27-29) and 5,30 (fractions 41-43)

Benefits:

• no polymer added, this protocol is suitable for the purification of proteins and protein complexes prior to crystallization
• no commercial ampholytes used, this protocol gives first chance to analyze isoforms for clinical applications

example 2:

isoform separation of Ovalbumin, isoforms between pH 4,5 - 4,7

Benefits:

• no polymer added, this protocol is suitable for the purification of proteins and protein complexes prior to crystallization
• no commercial ampholytes used, this protocol gives first chance to analyze isoforms for clinical applications

example 3:

isoform separation of Amyloglucosidase, isoforms between pH 3,6 - 3,9

Benefits:

• no polymer added, this protocol is suitable for the purification of proteins and protein complexes prior to crystallization
• no commercial ampholytes used, this protocol gives first chance to analyze isoforms for clinical applications

2. Very high throughput for denaturing separations.

2.a) Quick denaturing prefractionation, low resolution

pH gradient of the fast separation of human thyroid cancer cells under denaturing conditions between pH 3 - 10, separation buffer only used at one inlet to simulate a small separation chamber for quick prefractionation. FFE conditions Glycerol-HPMC buffer pH 3-10, transit time 64 Seconds

Fast separation of human thyroid cancer cells under denaturing conditions
First dimension FFE-IEF, second dimension SDS-PAGE

2.b) Quick denaturing prefractionation, medium resolution

Fast separation of human thyroid cancer cells under denaturing conditions with higher resolution
First dimension FFE-IEF, second dimension SDS-PAGE

2.c) Quick denaturing prefractionation, high resolution, buffers without polymer

Fast separation of human thyroid cancer cells under denaturing conditions in Urea - Mannitol buffers with high resolution. First dimension FFE-IEF, second dimension SDS-PAGE, FFE conditions Urea-Mannitol buffer pH 3-10, transit time 3.5 Minutes.

3. New protocols for protein complex separation

3.a) Native IEF separation of protein complexes

Native IEF separation of DPE2 protein complex, first dimension IEF FFE, second dimension
SDS-PAGE

3.b) Native electrophoretic sieving for separation and purification of Protein Complexes

Electrophoretic sieving is a three step process to enrich the protein complex first in a separation buffer with low concentration of polymers then on a second step a high resolution separation is performed at a higher level of polymer. Finally the polymer is removed by ITP

First step, EM separation with low concentration of Polymer

Second step, sieving with high concentration of Polymer

Sieving complete workflow

I) Separation according to EM  of Rubisco sample spiked with Amyloglucosidase and Trypsin inhibitor, first step low concenbtration of polymer, visualization SDS-PAGE

4. Customized ultra flat pH gradients with Ampholines for dedicated separation needs

Fractionation of raw ampholines covering pH 3-9.5 in the fist step, then using e.g. the cut between 7.5 - 8.5 for fine separation

Ultra flat IEF fine separation with custom ampholine gradient between pH 7.5-8.5

5. Reproducibility and recovery

Recovery rate

FFE is a loss free technology, everything which enters the separation chamber can be collected after sepraration, because no solid matrices like gels or columns are used. This enables highest recovery rates