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Department of Molecular Physiology

Our interests

biochemistry
2D - gel electrophoresis
Astacin - proteases (under construction)

The ciliary membrane subproteome of olfactory sensory neurons consists in the broader sense of three major protein groups. Integral membrane proteins, membrane associated proteins and their interaction partners comprise proteins involved in the olfactory signal transduction pathway as well as odorant metabolizing enzymes.

In our work we are mainly focused on integral membrane proteins such as ion channels participating in olfactory signal transduction. To get a more detailed insight into the structure, activity, spatial distribution, quantity and molecular interactions we use different two dimensional gel electrophoresis techniques for a high resolution purification and in a further step systematic identification of ciliary membrane proteins.

By 2D-IEF/ SDS-PAGE proteins are separated according to their charge (pI) by isoelectric focusing (IEF) in the first dimension and according to their size by SDS-PAGE in the second dimension. The IEF-method determines the isoelectric point (pI) of a protein by carrying out electrophoresis in a gel containing a pH gradient (IPG-strip in most cases). At a given pH proteins applied to this gel will be either positively or negatively charged. Upon the application of a current, a charged protein will migrate towards the anode or cathode to the part of the gel which corresponds to its isoelectric point (pI). At this point a protein has no net charge and will therefore stop migration. Finally all proteins are immobilized in the IPG strip which is then placed on an SDS-gel for second dimension electrophoresis. This procedure offers a unique capacity for the resolution of complex protein mixtures, permitting the simultaneous analysis of hundreds or even thousands of proteins. However, due to their broad hydrophobic regions and therefore poor solubility in aequeous solutions, membrane proteins are difficult to access via classical approaches like 2D-IEF/ SDS-PAGE and thus are notoriously under-represented in 2D-IEF/ SDS gels. In addition membrane proteins often precipitate around their mostly basic isoelectric points. One of our major goals  therefore is to modify this technique by the use of an ideal composition of different detergents  and chaotropic salts which allows to keep membrane proteins in solution.

2D-CTAB/ SDS-PAGE is a two dimensional gel electrophoresis technique that is especially suited to separate membrane proteins. Even those proteins with strong basic pI are displayed. Separation occurs in both dimensions according to size. CTAB is a cationic detergent which stabilizes positive charges outside the hydrophobic regions of membrane proteins. This helps to keep them in solution. The stabilizing effect is supported by an acidic pH. In addition to this effect, acid conditions promote partial removal of soluble proteins.

By Blue Native-PAGE (BN-PAGE) proteins are separated under native conditions. This offers the opportunity to maintain whole protein complexes. This technique uses Coomassie G250 as substitute for ionic detergents which is able to bind on the surface of proteins thereby inducing a negative charge shift. Non ionic detergents like Triton X-100, β-dodecyl maltoside or digitonine help to increase protein solubility while Coomassie G250 prevents agglomeration and precipitation due to charge repulsion effects. Combined with SDS-PAGE in the second dimension the subunit composition of a given complex can be determined and interaction partners can be identified.

 


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