4 replies to this topic

[dysbindin]

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?

[dysbindin]

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

[zogene]

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

[dysbindin]

zogene, on 16 July 2012 - 10:42 AM, said:

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

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

[zogene]

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.