Hi, not really sure based on your explanation. Probably your TF have intrinsic TAD? Do you put this bhlh79 in BD (i.e pGBKT7) and combine it with control (i.e empty AD; pGADT7)? Do you try reverse combination?

My guess is that your BHLH (being a transcription factor itself) is activating transcription of the His gene in yeast. I agree with Hendry Susila about a few control tests to check for activity of your BHLH by itself in the system.

Hendry Susila

Hi

I apologize for not being able to fully explain the process to you

I agree with you that maybe my TF has TAD

Yes, I connect my gene BD vector.

Yes, I combined it with control pGADT7.

And I use a +CONTROL and -CONTROL

Can I use 3-AT

because 3AT is a competitive inhibitor used as a reporter gene in Y2H

Hi Syed Sohail Ahmad

Yes, you can try using 3AT; at some concentrations, you might not see any growth in TDO/QDO. Apologies, I just saw your question title. Do you want to see the location of TAD? This webserver might be helpful: https://www.med.muni.cz/9aaTAD/, although it needs to be verified using truncation analysis.

I am not sure what your plan is, but if you want to perform a one-to-one interaction, it would be better to put your bhlh in the pGADT7 vector and the other target in the BD vector. Good luck!

Adding 3AT is a good choice, you'll need to look at possible levels, I think I used 7.5 mM myself. Used cultures diluted to OD 0.02 and 10 ul droplets, scanned plates at 7 days growth. The 3-AT will slow down strain growth in general so don't be surprised if you need more incubation days to see robust growth on your spot plates.

You could also clone domains of your protein into the system, that way you could find out which parts of your BHLH interact with your target proteins.

Hendry Susila Thanks

Professor, I am starting my work on cold stress in chili peppers. My work is based on upstream regulators, interacting proteins, and target genes and studies the regulatory network of the gene(bhlh-79).

Amy Klocko

Thanks, Professor.

• Your guidance will be very helpful to me in my work.

Amy Klocko Hendry Susila for 100ml how much 3-AT do we use?