Common Problems and Solutions for Yeast Two-hybrid Experiments-Yeast Display Technology Special Topic-Tekbiotech-Yeast Display Service,Phage display technology

Common Problems and Solutions for Yeast Two-hybrid Experiments

1. How to deal with the situation that some proteins may be difficult to analyze by two-hybrid due to their sequence characteristics?

Answer:

The yeast two-hybrid technique has a certain selectivity for protein sequences, which may lead to the failure to detect certain interactions. To overcome this problem, other connected experimental methods can be considered, such as co-precipitation experiments to confirm the interaction.

2. What are the commonly used plasmids for the GAL4 two-hybrid system?

Answer:

The commonly used vectors are pGBKT7 and pGADT7. Both vectors are shuttle vectors that contain replication start sites for E. coli and yeast, and contain different resistance and amino acid defect screening markers.

3. What are the main uses of yeast two-hybrid?

Answer:

Discover proteins that interact with known proteins (usually achieved by screening libraries); confirm the interaction between two proteins; identify regions involved in protein interactions.

4. What are the commonly used reporter genes for self-activation detection?

Answer:

The self-activation of the bait is detected by the expression of the reporter gene. Two-hybrid reporter genes include HIS3, ADE2, MEL1, lacZ and AbAr. Theoretically, all reporter genes can be used for self-activation detection. However, the reporter genes commonly used for two-hybrid self-activation detection are only AbAr+MEL1 combination or HIS3. With existing experimental techniques, self-activation produced by AbAr and HIS3 is more likely to obtain corresponding inhibitors.

5. How to deal with false negatives in yeast two-hybrid experiments?

Answer:

If the experimental results show that proteins that are known to interact are not detected during the experiment, it may be caused by factors such as incomplete protein folding or local structure, low expression, and lack of transcriptional activators. Using culture media under different conditions, optimizing protein expression levels, and choosing appropriate culture time and temperature may be able to solve this problem.

6. How to deal with false positives in yeast two-hybrid experiments?

Answer:

False positive results may be caused by non-specific or irrelevant protein interactions. In order to reduce false positive results, a series of countermeasures can be taken, such as adding negative control groups, using multiple protein tags, and performing sequence analysis and functional verification.

7. What should I do if there are multiple fragments in the obtained positive clones by PCR detection?

Answer:

It may be because a yeast cell can contain multiple capture proteins. You can try to select a single clone and streak it 2-3 times for blue-white screening until there is no separation and find a positive clone for subsequent experiments.

8. What should I do if there are too many clones on the screening medium?

Answer:

It may be that the bait protein is self-activated and can activate the expression of downstream screening markers by itself; or the screening conditions are too loose. You can use more stringent screening conditions to inhibit the self-activation activity of the bait protein. If the effect is not ideal, you can delete the domain of the bait protein that can cause self-activation activity and then use it in the yeast two-hybrid experiment.

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