Optimierung von Elektrophorese- und Blottingtechnik der vWF-Multimeranalyse

Doctoral thesis German OPEN
Thiel, Christopher (2010)
  • Subject: Agargel-Elektrophorese | Blotting | Immunoblot | Willebrand-Jürgens-Syndrom
    • ddc: ddc:610

Die vWF-Multimeranalyse ist in ihrem Ablauf eine sehr komplexe, arbeitsaufwendige und störungsanfällige Methode zur Diagnostik eines VWS. So war es in der Vergangenheit schwierig, einen streng horizontalen Lauf der Plasmen während der Elektrophorese sicherzustellen. Es kam oftmals, vermutlich durch Leitfähigkeitsunterschiede, zu einem Aufstieg und Übertritt der Plasmen in den darüberliegenden Elektrodenpuffer. Ausserdem war es notwendig vor dem Blotten einen exakten Kontakt zwischen Agarosegel und Nitrozellulose herzustellen. Ansonsten kam es, bedingt z.B. durch Luftblasen, zu einem unvollständigen Transfer der Multimere vom Gel auf die Nitrozellulose. In diesem Fall war die nachfolgende Ausbildung eines sauberen Bandenmusters nahezu unmöglich. In dieser Dissertation sollen zwei Veränderungen in der Vorgehensweise der vWF-Multimeranalyse beschrieben werden, die zu einer höheren Stabilität und Robustheit dieser Methode beigetragen haben. Dadurch konnte ein klar abgrenzbares Bandenmuster erhalten und somit die Diagnostik eines VWS erleichtert und verbessert werden The high-resolution imaging of vWF multimers with main- and accessory bands over the extreme range of protein size from 556 kDa homodimer to over 20,000 kDa multimers requires particular optimization of the electrophoresis technique. Only few laboratories are skilled to perform this analysis. Most critical points of MA (vWF-Multimer analysis) are to achieve an undisturbed electrophoretical run over a relatively long distance necessary to divide the single bands of heavy multimers and to display the triplett (or quintuplett) structure of the smaller multimers. Furthermore, during blotting an unimpeded transfer of these fine bands must be ensured. Sometimes the analyte may escape from the gel into the covering electrode buffer. Possibly, this is caused by a leakage current which may result from the higher conductivity of the electrode buffer compared to the separating gel. We obtained an undisturbed run through the separating gel by topping it with an insulating agarose gel layer produced by casting 1% agarose solution in deionized water on top of the stacking and separating gels. The most precarious step in blotting procedure is to adhere the membrane strictly to the gel: Trapping of airbubbles has to be avoided. No space between gel and paper must remain, although no pressure must be exerted to the gels, because agarose gels are not cross-linked and consequently very pressure-sensitive. To elude this problem, we sticked the membrane with agarose (0.8% in separating gel buffer) onto the separating gel, avoiding inclusion of airbubbles. Thus a direct contact of agarose to the blotting membrane could be achieved without any pressure. In addition we tested the so called semi-dry blotting-system and compared it to the tankblotter. As a result, this tested blotting-system is not so expensive like the tankblotter, but we got results of it, which helped us to find easier the diagnose.
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