the membrane system yeast two-hybrid vector is selected according to the different positioning of the bait protein, which is beneficial to the interaction between ubiquitin NubG and Cub-LexA-VP16 linked to the interacting protein, and is recognized by UBPs, thus starting the expression of the downstream reporter gene. So what carriers are there in the membrane system? How to choose specifically? Xiao Zheng is giving you some advice.

1. What bait carriers are there in the membrane system?

there are three kinds of bait carriers for membrane yeast two-hybrid system, which are as follows:

pbt3-n

pbt3-suc

pbt3-ste

2. How to choose the carrier?

according to the different protein domains, the selected vectors are different. In the following table, we summarize the vectors suitable for different situations.

so how to predict the protein domain? The specific prediction process is as follows:

1. Translation of CDS sequence into amino acid sequence

/tools/translate.html

2. Protein transmembrane domain prediction

https://services.healthtech.dtu.dk/service.php? TMHMM-2.

3. Protein signal peptide prediction < P > https://services.healthtech.dtu.dk/service.php? SignalP-5.

4. Protein domain prediction

http://smart.embl-heidelberg.de/

3. Delivery standard of membrane system sieve library?

what kind of membrane system screening results are qualified? Xiaozheng takes you to intuitively experience the payment standard of membrane system screening library. Taking the screening of membrane system library with pBT3-STE as an example, * * * counts seven steps and seven conclusions.

1. Construction of bait vector

Conclusion 1: Whether the vector is correct or not is compared by first-generation sequencing.

2. Functional verification of bait vector

Conclusion 2: Whether the constructed bait vector has frameshift mutation, whether the ubiquitin-specific protease system can function normally, and whether to carry out subsequent screening experiments. The results show that the follow-up screening test can be carried out.

3. Self-activation and toxicity detection

Conclusion 3: Whether the bait carrier pBT3-STE-XXX has self-activation or not, and whether it can be used for subsequent screening experiments. The results show that the follow-up screening test can be carried out.

4. Primary screening of yeast library

Conclusion 4: How many monoclonal antibodies were screened by primary screening.

5. Re-screening the primary screening results

Conclusion 5: How many positive clones were screened by re-screening.

6. rotary verification

conclusion 6: the yeast positive clone plasmid was rotary verified to determine how many true positive genes there were.

7. Point board verification

Conclusion 7: (Personalized display can be made according to the requirements of the article) Determine whether it is consistent with the rotary verification results.