optimising yeast two hybrid testing conditions

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optimising yeast two hybrid testing conditions

Post by fcs3 » Mon Jul 25, 2011 7:58 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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Inland Taipan
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Post by JackBean » Sat Jul 30, 2011 6:09 pm

1) yest don't grow at higher-than-30°C temperatures, so be sure, you measure your temperature accurately ;)
2) if your plant grows at 20°C, that might be a problem. Try it and you will see, what will you get ;)

Cis or trans? That's what matters.

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