Transfection and analysis. ori

Transkript

1 Functional promotor?? Transfection and analysis ori

2 luciferin vpu ORF env ORF controle in Tausend Luciferase activity x 10-3

3 Promotor-Deletionsanalyse

4 Figure DNase I footprinting identifies protein-binding regions in a DNA molecule by their ability to confer resistance to cleavage by DNase I

5 ? DNA bindendes Protein? DNA Bindungsstelle? synthetisches doppelsträngiges Oligonukleotid PCR Markierung Markierung

6 Gel shift assay oder Electrophoretic mobility shift assay (EMSA) Überprüfung der Identität des gebundenen Proteins? Antikörper Protein markiertes Fragment nicht-markiert Polyacrylamidgel (nicht denaturierend??)

7 Die Erkennung des Proteins im Protein-DNA Komplex durch den Antikörper führt zu einer langsameren Laufgeschwindigkeit (Supershift) Antikörper Protein markiertes Fragment nicht-markiert Markiertes Fragment Kontrolle Fragment + Protein (Shift) Komptetition mit unmarkiertem Fragment Fragment + Protein + Antikörper (Supershift)

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9 Wie kann man Protein-DNA Wechselwirkungen direkt beobachten und kann man die Dynamik solcher Prozesse untersuchen? McNally J et al, Science, 200

10 McNally J et al, Science, 2000

11 Fig. 3. GFP-GR binding to the MMTV LTR in living cells cells (8) were grown in Dulbeco Õs modiþed EagleÕs medium (DMEM) (Gibco BRL, Grand Island, NY ) supplemented with 10% fetal bovine serum (Hy-Clone, Logan, UT ) in the presence of tetracycline (5 mg/ml). For GFP-GR imaging, cells were transferred either to 22-mmsquare cover slips resting in wells of a six-well plate or to selfenclosed chambered cover slips (Nalge Nunc International, Naperville, IL). At the time of transfer, the medium was replaced with DMEM without tetracycline (to induce GFP-GR expression) and supplemented with 10% charcoal-dextranðtreated serum (to prevent hormone stimulation of the GFP-GR). After 16 to 24 hours, dexamethasone was added to the cells to induce nuclear accumulation of the GFP-GR. After 2 hours of dexamethasone treatment, cells were imaged with a 1003/1.4 numerical aperture oil immersion lens on a Leica TCS NT SP laser scanning confocal microscope. GFP was excited with the 488-nm line from an argon laser. The confocal pinhole was set at 1.0 Airy disk unit. (A) A two- imensional (2D) section showing a representative nucleus after 2-hour induction with 5 nm dexamethasone. Arrow indicates the array. (B and C) Projections of 3D images of two nuclei from the same Þeld, each showing a single array structure (arrows) after 2-hour treatment with 1 nm dexamethasone. Scale bars, 5 mm. McNally J et al, Science, 2000

12 Ko-Lokalisierung von ras-spezifischer nascierender RNA und GR-GFP Fusionsprotein Mit Photo Bleach kann die Dynamik der Bindung gezeigt werden McNally J et al, Science, 2000

13 Protein-Protein Wechselwirkungen

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15 Untersuchung von Protein-Protein Interaktionen Reportergen DBD DNA binding domain TAD Transactivator domain

16 DBD DNA binding domain TAD Transactivator domain

17 ? Protein 1 Protein 2

18 Protein 1 Protein 2 DBD DNA binding domain TAD Transactivator domain bait prey

19 Reportergen bait prey

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25 Gibt es Einschränkungen bei der Auswahl der Proteine? Mit Yeast 2 Hybrid and Bakterien 2 Hybrid können nur Interaktionen zwischen löslichen Proteinen untersucht werden Transiente Wechselwirkungen sind kaum zu detektieren Wie Untersucht man Wechselwirkugen zwischen Membranproteinen?

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27 Ubiquitin-speci protease

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34 Wurde die Interaktion zwischen zwei Proteinen in einem Screening System identifiziert, muss anschließend die Spezifität der Interaktion überprüft werden!? Echt oder Artefakt? Echte Interaktion Artefakt Molekulargewichtsmarker

35 Weitere Verfahren zur Untersuchung von Protein-Interaktionen

36 Analytical versus functional protein microarrays. a, Analytical protein microarray. Different types of ligands, including antibodies, antigens, DNA or RNA aptamers, carbohydrates or small molecules, with high affinity and specificity, are spotted down onto a derivatized surface. These chips can be used for monitoring protein expression level, protein profiling and clinical diagnostics. Similar to the procedure in DNA microarray experiments, protein samples from two biological states to be compared are separately labelled with red or green fluorescent dyes, mixed, and incubated with the chips. Spots in red or green colour identify an excess of proteins from one state over the other. b, Functional protein microarray. Native proteins or peptides are individuall purified or synthesized using high-throughput approaches and arrayed onto suitable surface to form the functional protein microarrays. These chips are used to analyse protein activities, binding properties and post-translational modifications. With the proper detection method, functional protein microarrays can be used to identify the substrates of enzymes of interest. Consequently, this class of chips is particularly useful in drug and drug-targ identification and in building biological networks. Phizicky E, et al. Nature, 2

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38 Abb. 2: Sensorgramm. Die Kästchen geben den Zustand amsensor Chip und das Diagramm die Anzeige ambildschirm wieder. a) Basislinie impufferfluss b) Probeninjektion, Bindung und Signalanstieg c) Dissoziation und Signalabfall im anschließenden Pufferfluss d) Basislinie nach der Regeneration

39 Mit SPR lässt sich die Bindung zwischen 2 Partnern sudieren SPR bietet aber auch die Möglichkeit, einzelne Substanzen schnell und sensitiv nachzuweisen

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53 Fragen? Anregungen?