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Please inform us if there are any follow up actions we need to talk with the members of the congress : |
I do not know.But I will try to register our matrix:
Specific primers list:
Laminin,Fibronectin ,collagen type 1, ,collagen type 4 , Desmin, Actin, GAPDH, ITGA5, ITGBL1
In recent years, separation of protein complexes by blue native polyacrylamide gel electrophoresis (BN-PAGE) has proven to be a powerful method for identifi cation of functional disorders in total tissue homogenates, cells and cell fractions [1–4]. The method has been used to determine the molecular weight of protein complexes in the range between 10 and 10 000 kDa.Especially the membrane intrinsic electron/proton transfer complexes
in mitochondria and chloroplasts have been analyzed with success .The method has been combined with other techniques to determine the oligomeric state, stochiometry, enzymatic activity and the molecular structure
of multiprotein complexes.As many samples may be separated in parallel in one electrophoretic run, a direct comparison of protein complexes readily allows the identify cation of disorders and direct further functional
analysis. In proteomics, high resolution separation of proteins is generally achieved by two-dimensional (2-D) separation according to the proteins isoelectric point (IEF-PAGE) and molecular mass (SDS-PAGE). This denaturing technique separates hundreds to thousands of proteins in complex samples [11]. However, hydrophobic membrane proteins are hardly detectable after 2-D IEF/SDS-PAGE [12]. Therefore, global analysis of membrane proteomes has not been started, despite the importance of membranes for the living cell.Here, BN-PAGE presents an alternative strategy to separating membrane proteins with high resolution and maintaining their enzymatic function [13]. The method is called native, as most separated protein complexes retain enzymatic functions and blue native, since electrophoretic separation relies on binding of the dye Coomassie blue G250 to
protein. Blue native PAGE was fi rst described in 1991 for the separation of membrane protein complexes from the respiratory chain of human mitochondria.Today, BN-PAGE is well-established as an excellent choice
when analysis of protein interactions between native membrane proteins is required at high resolution and high throughput [5,15]. But why is it of importance in biology to know about protein interactions?
Proteins express the function of genes in all organisms that exist on earth. Proteins are the functional molecules which operate metabolic, developmental and regulatory pathways in a cell, tissue or an organism. Multiple proteins are integrated in complexes in order to organize multi-enzymatic functions. Protein complexes therefore refl ect the cell’s macromolecular organization state which is a fundamental basis for understanding cellular functions. In practical proteomics, the goal is to identify all proteins present in a defined developmental state of an organism, tissue, cell or their subfractions and to characterize their qualitative and quantitative changes in response to environmental changes [17–19]. Although protocols have been worked out in general to start proteomic analysis in the many domains of life, systematic proteome analysis awaits the development of adequate technology. In this respect, BN-PAGE is one of the candidates to standardize the first step during the sequential high resolution analytical fractionation of the proteomes [20]. In particular, the natural organization of living systems has offered this opportunity to trace the function of native protein complexes after biochemical
separation in BN-PAGE. Due to the association of a large number of single proteins in multi-enzymatic protein complexes, the level of complexity is maintained at a low level. This is the basis for every successful technological analysis of dynamic developmental and metabolic states and organization levels. With BN-PAGE, a simple technology for separation and identifi cation of protein complexes, and especially of membrane protein complexes, has been established for a high number of developmental states over recent years. In the following, allow me to explain the principles of BN-PAGE and show you how to get started with the technology.
Materials
Sample Preparation for BN-PAGE
1. Sample buffer BN: 750 mM - aminocaproic acid, 50 mM Bis-Tris-HCl pH 7.0, 0.5 mM EDTA-Na2.
2. Detergent solution: Dodecylmaltoside solubilization buffer: 10 % (w/v) n-dodecyl--D-maltoside.
Digitonin solubilization buffer: 30 mM HEPES pH 7.4, 150 mM potassium acetate, 10 % (v/v) glycerol,
5 % (w/v) Digitonin.
3. Loading buffer: 750 mM -aminocaproic acid, 5 % (w/v) Coomassie G 250.
Casting of Gradient Gels for BN-PAGE
1. BN gel buffer (6 ×): 3 M -aminocaproic
acid, 0.3 M Bis-Tris-HCl pH 7.0. 2. Acrylamide solution: 30% (w/v) acrylamide/
bis acrylamide solution (37.5:1, 2.6%C).
3. Glycerol (100%).
4. TEMED: N,N,N,N`-tetramethyl-ethylenediamine.
5. APS: ammonium persulfate:
10% (w/v) solution.
6.Water-saturated isobutanol:
50% (v/v) isobutanol.
BN-Electrophoresis
1. BN running buffer cathode (10 x): a. Blue cathode buffer: 500 mM Tricine, 150 mM Bis-Tris-HCl pH 7.0, 0.2 % Coomassie G250.
b. Colorless cathode buffer: 500 mM Tricine, 150 mM Bis-Tris-HCl pH 7.0.
2. BN running buffer anode (10 x): 500 mM Bis-Tris-HCl pH 7.0.
Solubilization and Transfer of BN Strips
1. BN solubilization buffer: 2% (w/v) SDS, 66 mM Na2CO3, 0.67% - Mercaptoethanol.
2. Overlay solution: 0.5% (w/v) agarose in 1 × SDS running buffer.
Casting of Second Dimension Gels
1. SDS separating gel buffer (8 x): 3 M Tris pH 8.8.
2. SDS stacking gel buffer (2 x): 250 mM Tris pH 6.8.
3. Acrylamide solution: 30% (w/v) acrylamide/ bis acrylamide solution (37.5:1, 2.6%C).
4. TEMED: N,N,N,N`-tetramethyl-ethylenediamine.
5. APS: ammonium persulfate: 10% (w/v) solution.
6.Water-saturated isobutanol: 50% (v/v) isobutanol.
SDS Electrophoresis
1. SDS running buffer (10 ×): 250 mM Tris,
1.92 M glycine, 1% (w/v) SDS.
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Please give a briefing of your own observations and outcomes of the congress: : |
Brief report of participation in ECCO17,ESMO38:It is an International congress had been hold in Amsterdam from September,27 to First of October 2013 this year.The thing that seemed interesting to me was cooperation and harmony between different oncology groups from surgical to medical ,radiation to radiotherapy,epidemiology to ethics commities in this great meeting.The slogan of congress was reinforcing multidisciplinarity.They did it very well.There was variety of sessions such as teaching lectures,mentorship sessions,preffered papers,clinical debates and challenging problems,translational researches,society sessions,keynote lectures,..and poster discussions.I liked advocacy and ethics about best practices.Case based discussions was very informative to me as well.My poster had been discussed afternoon of third day of congress after presentation in the morning.There was some dout about structure and composition of human fat tissue scafold I presented.I explained about proteomic study we had done after sending abstract.That complementery work was based on Western-Blot and RT-PCR studies. |