Help on the yeast two hybrid - (Jul/09/2012 )

Hi everyone,

I am trying to use yeast two hybrid system to screen some interaction proteins. But I have big problem on the bait expreesion. I cloned the full length cDNA with start coden into pGBKT7 vector. The transformed yeast with this plasmid could growth on the -Leu SD plat, which means the vector were transformed into yeast strain. However, I can not detect the any expression bait by western, wheras, the pGBKT7-p53 could be detect. I am sure that my cloning work is right , as the sequencing shows no frame shit happened. Does anyone met this problem before? Can I use this bait to continue my screening?

Holy...It seems no one else here met this problem

Hello

sorry I saw your post just today, I did Y2H screening using same vector, first regarding to grow in SD/-Leu did you do the experiment once or couple times, which strain did you use, what is the genotype of it, I will suggest go back to your original stock streak again and use fresh clone but why did you use SD/-Leu any way it is for prey not bait, secondly regarding to expression issue did you use c-Myc Ab or specific Ab for your gene and is you gene in the frame or not this is so important?

regards,
zogene

zogene on Mon Jul 16 18:42:14 2012 said:

Thanks! Sorry for seeing your reply so late, and regarding your questions, I indeed use fresh clone which could be growth in SD/-Leu, and I do this several times. The expression vector is in frame due to our sequencing.
I also test the expression in BL-21 E.coli as the pGBKT7 has T7 promter. However, in BL21, I can get very strong and special western band detected by myc antibody in the right molecular weight. Is there any possibilty that this protein is regulated tightly in yeast AH109 , so that we can not dectect ? ( the cDNA i insert in vector is yeast DNA).


Best

dysbindin

Hi

Again, why did you plate them on SD/-Leu instead of SD/-Trp, regarding to western blotting expression level may vary from insert to another, you may have very week band I repeated my western 5 times to get my band but it was week but with my gene specific Ab it was very clear and strong? so I would not advice to proceed your experiment till you get your band in western, and read Clontech yeast handbook it is very useful.

good luck.