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yeast two hybrid optimising conditions


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#1 fcs

fcs

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Posted 25 July 2011 - 12:27 AM

Hi All,

Does anyone know anything about changing growth temperature for yeast two hybrid analysis to ensure temperature is appropriate for protein conformation? I'm looking at interactions between full length plant flowering proteins that are orthologous to proteins shown to interact in 3 other diverse angiosperms (monocots + dicots) - so I would be surprised if they didn't interact in my species, but at standard yeast growth conditions of 30'C (manufacturer's instructions) I get no evidence of interaction from -HIS+3AT, Xgal assay, -URA. I've read articles where people have done yeast two hybrid tests at 25'C or 22'C to ensure appropriate temperature for protein conformation.
Are these temperature choices usually based on optimal growth conditions for the plant species the proteins studied are from?
Is it sensible to test interactions at 2O'C if my plant species grows best at this temperature?
Does anyone have experience with optimising yeast two hybrid conditions to detect interactions?
Any other ideas of things I could try?

Many thanks




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