Modified
Dec 2012
RW

 

FFE_01

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

Elastase_9_11_gradient

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

Elastase

M= protein marker
S= crude sample
66-79= FFE Fractions

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


OVA_gradient

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

Ova1

M= protein marker
S= crude sample
40-52= FFE Fractions

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

isoform_grad

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

isoform_gel1

M/S= protein marker = used as sample

19- 53= FFE Fractions
Am = Amyloglucosidase
GO = Glucose Oxidase
TI = Trypsin Inhibitor

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


3. Separation of Isoforms (new protocol)

Microsoft PowerPoint - [IEF0922_6369p4

 

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
Microsoft PowerPoint - [IEF0929_6379p8

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

 

Microsoft PowerPoint - [IEF0922_6369p7

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

HT-CE_grad

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

HT-CE_1

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

HT-CE_2_grad

pH gradient of the fast separation of human thyroid cancer cells under denaturing conditions between pH 3 - 10, separation buffer only used at two  inlets to simulate a small separation chamber for quicker prefractionation. FFE conditions Glycerol-HPMC buffer pH 3-10, transit time2 Minutes

HT-CE_2

M= protein marker
S= crude sample
17-44= FFE Fractions
 

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

HT-CE_3_grad

pH gradient of the fast separation of human thyroid cancer cells under denaturing conditions between pH 3 - 10, separation buffer only used at three inlets to simulate a small separation chamber for quicker prefractionation.

HT-CE_3

M= protein marker
S= crude sample
19-53= FFE Fractions

Unlimited concentraion possible after FFE separation
 

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

DPE_2_1

M= protein marker
S= crude sample
31-38= FFE Fractions


 

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

sieving1+3

Second step, sieving with high concentration of Polymer

sieving2+1

Sieving complete workflow

sieving3+4

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

Rubisco3

M= protein marker
S= crude sample
25-56= FFE Fractions
Am= Amyloglucosidase
TI= Trypsin Inhibitor

 

II) Electrophoretic sieving of Rubisco sample spiked with Amyloglucosidase and Trypsin inhibitor, second step high concentration of polymer, visualization SDS-PAGE

Rubisco4

III) Third step ITP enrichment of Rubisco to remove the polymer,
visualization SDS-PAGE

Rubisco_ITP4

M= protein marker
S= crude sample
3 -40 = FFE Fractions
 

FFE conditions:

ITP to remove polymer
 

M= protein marker
S= crude sample
47-77= FFE Fractions
Am= Amyloglucosidase
TI= Trypsin Inhibitor

FFE conditions:

sieving with high concentration of polymer
 


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

amph_sep1

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

amph_sep2

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


5. Reproducibility and recovery

repro1

Fast denaturing separation of six samples of human thyroid cancer cells under denaturing conditions
First dimension FFE-IEF, second dimension SDS-PAGE, CBB staining (blue silver)

FFE conditions
Glycerol-HPMC buffer pH 3-10
transit time 2 Minutes
 


repro2
repro32

Recovery rate

recover2

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