Participant from IBIR: Alexander Kukov

The name of the visited institution: Interuniversitäre Department für Agrarbiotechnologie, IFA Tulln, Austria Department IFA-Tulln, Institute of Biotechnology in Animal Production. http://www.ifa-tulln.ac.at/ Head of the Laboratory is Prof. Dr. Urban Besenfelder, ReProForce project partner.

Duration: 06.05.2012 – 13.05. 2012.

Purpose of the visit:
The purpose of this visit was the introduction into methods for investigation of molecular changes in the reproductive organs in female animals after insemination.

The research program included:
The visited research group of the Department focuses its work on protein analyses of oviduct endothelial cells. The laboratory is well equipped with apparatus for proteins investigations: SDS PAGE, isoelectric focusing (IEF), two dimensional electrophoresis (2D), densitometry scanners etc. Mass spectrometry analyses are carried out in another laboratory in Veterinary Medicine University, Vienna.
Investigation of the spermatozoa effect on oviduct endothelial cells after insemination was done. For this purpose, phosphoprotein synthesis from rabbit oviduct endothelial cells during insemination was analysed by 2D electrophoresis and other approaches. Phosphoproteins from cell lysates were isolated and stained with fluorochrome dyes Cy 3 and Cy5, then isoelectrical focusing of proteins was performed. Dry strips (BioRad) were rehydrated for 12 hours, after that IEF was launched. Further the strips were incubated in DTT for 20 min, IAA for 20 min and then subjected to second electrophoresis using Precast Gels (BioRad) for proteins separation by molecular weight. The gels were scanned and subjected to silver staining. For silver staining, the usage of glutaraldehyde was deliberately avoided because some spots were further analysed by MALDI TOF. A short training on MS-FIT, MS Taq, ExPASy software was held. These programs were used to analyze the obtained mass spectrometry data.

Conclusion: The planned program was successfully completed. During the work in the laboratory I have trained basic approaches of IEF of proteins, fluorochrome dyes labeling of proteins and scanning of gels.