Modified
Dec 2012
RW

 

FFE_01

Advances in Free Flow Electrophorsis since 35 years

 Existing applications of Free Flow Electrophoresis

 PROTEINS

 Proteomes from:
 human tissue
 body fluids
 E.coli
 yeast
 plant tissue

 Isoforms of:
 glycoproteins
 antibodies
 etc.

 MEMBRANES

 from:
human cells like:    
erythrocytes
thymocytes   
lymphocytes
kidney cells

plants

bacteria
 

 BIOPARTICLES

 bone marrow cells
 PMN leucocytes
 platelets
 thymus cells
 kidney cells
 spleen cells
 spermatozoa
 bacteria
 parasites
 etc.
 

 ORGANELLES

 melanosomes
 calciosomes
 endosomes
 lysosomes
 peroxisomes
 vesicles
 endoplasmic reticulum
 mitochondria
 etc.

 

 MISCELLANEOUS:
 chiral organic coumpounds, liposomes, peptide hormones, ions, nanotubes and nanoparticles

 Purification of mitochondria from Saccharomyces cerevisiae
 
Cooperation with Dr. Ueffing, GSF

The analysis of complex cellular proteomes by means of two-dimensional gel electrophoresis (2-DE) is significantly limited by the power of resolution of this technique. Although subcellular fractionation can be a fundamental first step to increase resolution, it frequently leads to preparations contaminated with other cellular structures. Here, we chose mitochondria of Saccharomyces cerevisiae to demonstrate that an integrated zone-electrophoretic purification step (ZE), with a free-flow electrophoresis device (FFE), can assist in overcoming this problem, while significantly improving their degree of purity. Whereas mitochondrial preparations isolated by means of differential centrifugation include a considerable degree of non-mitochondrial proteins (16%), this contamination could be effectually removed by the inclusion of a ZE-FFE purification step (2%). This higher degree of purity led to the identification of many more proteins from ZE-FFE purified mitochondrial protein extracts (n = 129), compared to mitochondrial protein extracts isolated by differential centrifugation (n = 80). Moreover, a marked decrease of degraded proteins was found in the ZE-FFE purified mitochondrial protein extracts. It is noteworthy that even at a low 2-DE resolution level, a four-fold higher number (17 versus 4) of presumably low abundance proteins could be identified in the ZE-FFE purified mitochondrial protein extracts. Therefore these results represent a feasible approach for an in-depth proteome analysis of mitochondria and possibly other organelles.

 Acidic Rat Liver ProteinsEnriched by IEF-FFE

Acidic rat liver proteins were separated by IEF-FFE prior to 2D-Electrophorsis. The fractionated samples show crucial enrichment of proteins compared to the unprocessed sample.

 Rat Liver Protein Separation by IEF-FFE

 High Resulotion Rat Liver Protein Separation by IEF-FFE

CBB stained gels of single fractions of FFE Separated Rat Liver proteins.
SDS-PAGE, T = 4-20%
 

CBB stained 2D-gels of single fractions of FFE Separated Rat Liver proteins.
First dimension IPG-IEF pH 3-10L, 11cm, second dimension SDS-PAGE, T=13%

==> ZOOMING in:
 

CBB stained 2D-gels of the single FFE fraction 49 (left) and the crude extract (right) of FFE Separated Rat Liver proteins. First dimension IPG-IEF pH 5-8, 11cm, second dimension SDS-PAGE, T=13%
 

 Identification of Integral Membrane Proteins from S. cerevisiae Mitochondria

 Membrane proteins from Corynebacterium glutamicum
 Cooperation with A. Burkovski (Cologne)

Silver stained SDS-PAGE of membrane proteins from Corynebacterium glutamicum

The separation was performed with standard Prolytes, urea, thiourea and mannitol.
No detergent was used during the FFE run.
 

 Identification of Integral Membrane Proteins from mitochondria of rat liver

 Enrichment of proteins from human plasma; Overview

 Enrichment of proteins from human plasma; CBB-stained 2D-PAGE

 SDS-PAGE of FFE-fractions after native depletion of human plasma

pH - profile of the FFE run from the native human plasma depletion

 SDS-PAGE of pooled FFE-fractions after Ultrafiltration (UF)

CBB - stained SDS-PAGE of depleted and FFE separated proteins from human plasma.

 Isolation of isoforms of proteins

 FFE - Enrichment of proteins from testis cells

 FFE Fraction        CBB-stained

 crude extract          CBB-stained

 FFE Fraction        CBB-stained

 crude extract          CBB-stained

 FFE Fraction        CBB-stained

 crude extract          CBB-stained

 FFE Fraction        CBB-stained

 crude extract          CBB-stained

The spot intensties of the Coomassie stained FFE Fractions are much higher than in the crude extract of testis cell proteins (see the marked red spot as example) although the protein concentration loaded to the gels was identical. We could observe also novel proteins in all FFE fractions (examples: yellow marked areas in the 3D image).