Příprava a testování polyklonálních protilátek proti Cbf11 a Cbf12, transkripčním faktorům rodiny CSL kvasinek Schizosaccharomyces pombe

• Thesis title in Czech: Příprava a testování polyklonálních protilátek proti Cbf11 a Cbf12, transkripčním faktorům rodiny CSL kvasinek Schizosaccharomyces pombe
• Thesis title in English: Preparation and testing of polyclonal antibodies against Cbf11 and Cbf12, the fission yeast CSL transcription factors
• Key words: proteiny CSL, Schizosaccharomyces pombe, heterologní exprese proteinů, afinitní chromatografie, příprava polyklonální protilátky
• English key words: CSL proteins, Schizosaccharomyces pombe, heterologous protein expression, immobilized metal affinity chromatography, polyclonal antibody preparation
• Academic year of topic announcement: 2012/2013
• Thesis type: Bachelor's thesis
• Thesis language: čeština
• Department: Department of Biochemistry (31-250)
• Supervisor: doc. RNDr. František Půta, CSc.
• Author: hidden - assigned by the advisor, waiting for guarantor's approval
• Date of registration: 03.01.2012
• Date of assignment: 17.12.2012
• Date of electronic submission: 27.05.2013
• Date of proceeded defence: 11.06.2013
• Opponents: RNDr. Michaela Čermáková, Ph.D.
• Advisors: prof. RNDr. Marie Stiborová, DrSc.

Preliminary scope of work in English

Cbf11 and Cbf12 are antagonistic paralogous proteins that are important for the proper coordination of cell and nuclear division in the fission yeast Schizosaccharomyces pombe. The expression levels of these two transcription factors need to be finely balanced, making it difficult to create fully functional tagged alleles with conventional knock-in strategies. Therefore, the availability of specific antibodies would greatly enhance the study of the CSL family in fission yeast.
Using bioinformatics, we will select suitable immunogenic Cbf11 and Cbf12 protein fragments, clone the corresponding DNA sequences, verify the constructs, and subclone them into a bacterial vector for his-tagged expression. We will optimize the expression and purification protocols for the tagged protein fragments, carry out large-scale protein purifications and submit the resulting material for commercial antibody production in rabbits.
We will validate the specificity of the polyclonal antibodies raised, and we will use them to determine the dynamics of Cbf11 and Cbf12 expression during the fission yeast cell-cycle.