Yeast two-hybrid technology and common problems

Yeast two-hybrid, proposed by Fields in 1989, is an experimental technique for studying the interactions of proteins in living cells under the eukaryotic model yeast.

This system can not only detect the interactions between known proteins, but also discover unknown proteins that interact with known proteins. It is widely used in animal and plant protein research and is a commonly used method in experiments. But at the same time, in the experiment, there will be various problems and no way to start. This article will sort out the common problems that may be encountered in the experiment, and hope it can be helpful to everyone.

1.After sterilization, the color of the yeast medium becomes tan.

SD series medium is premixed with glucose, which causes carbonization of glucose in the medium during the sterilization process due to a long time of sterilization or a long time of placement under high temperature conditions after sterilization, resulting in darker color.Under normal circumstances, the yeast culture will not have a great impact (for individual Pichia yeast species have a great impact), can be controlled by sterilization time and sterilization temperature to reduce the degree of carbonization, such as 118℃ sterilization for 15 minutes, and timely reverse plate.

2.Yeast medium does not gel, or the gel hardness is not enough.

The pH of yeast screening medium was adjusted (5.8).Appropriately increase AGAR (20g/L recommended).

3.Possible reasons why yeast cells turn pink during culture.

The concentration of adenine (ADE) in the medium was low.Or the metabolic pathways of yeast cells.Adenine sulfate (60mg/L) is usually added to the medium to improve the situation.

4.Yeast cells grow slowly.

Possible cause: The bait protein expressed by yeast is toxic to cells and affects the growth of yeast.

The solution: select low-sensitivity yeast strains; or use low-copy number expression vectors.

5.The bait protein is toxic to yeast cells. How to perform follow-up experiments?

Strains that do not grow well in liquid medium will grow well in solid medium.First, the clones were resuspended on 1 mL SD/ — Trp (PM2251), then resuspended on 5 100mm SD/ — Trp plates (PM2252) in a warm bath at 30℃ until the clones were bonded to each other on the plates.Clones were scraped off each plate with 5mL 0.5X YPDA (PM2011) and collected into a tube so that normal hybridization reactions could be performed using this cell resuspend.

6.Too many clones on the selection medium.

Possible reasons: There may be self-activation of the bait protein, which can activate the expression of downstream selection markers by itself; or the selection conditions are too loose.

Solution: select more strict screening conditions to inhibit the self-activation activity of bait protein, if the effect is not ideal, we can delete the structure domain that can cause the self-activation activity of bait protein, and then use it in yeast two-hybrid experiment.

7.Too few clones on the screening medium.

Possible reasons: yeast hybridization efficiency is low; or the screening conditions are too strict

Solution: prolonging hybridization time and improving hybridization efficiency;Relaxation of screening conditions.

8.The obtained positive clones have multiple fragments detected by PCR.

Possible cause: A yeast cell can contain multiple prey proteins.

Solution: select a monoclonal paddle for 2-3 times, carry out blue-white spot screening, until no separation, find a positive clone, for subsequent experiments.

9.Can X-α-gal only be dissolved in DMF?

In almost all yeast hybridization experiments, DMF is used to dissolve X-α-gal. X-α-gal is also soluble in methanol and DMSO. DMSO can be used for the preparation and dilution of X-α-gal in the experiments that verified the yeast hybridization. However, DMSO has a high melting point and is inconvenient to use. Methanol has not yet been verified.

10.What should I do if the hybridization efficiency is not high?

The efficiency is not high, possibly because the number of hybrid cells is not enough, and the liquid culture amount of bait protein should be appropriately increased to ensure the sufficient number of cells.The hybridization time can be prolonged until the presence of cloverleaf shaped zygote is observed by microscopic examination, and then the subsequent operation can be carried out.

11.In order to verify the interaction between two proteins when co-transferring AD to Y2HGold with BD, or should BD and AD transfer to Y2HGold together?

Both methods can be used. If AD is transferred to Y2H Gold with BD, use SD/-Trp medium to shake the bacteria when preparing the competent Y2H Gold strain containing BD vector.

12.Can I use sterile water instead of 0.9% NaCl solution to resuspend the bacteria after yeast transformation?

The purpose of resuspended with normal saline is to maintain osmotic pressure, so can yeast resuspended with sterile water. There is no significant difference in conversion efficiency between the resuspended with sterile water and the resuspended with normal saline.

13.How to reduce false positives in the yeast two-hybrid system?

Multiple screening markers can be selected for more stringent screening to reduce false positives;False positives can be reduced by increasing the concentration of ABA (CA2332) or 3-AT (CA1311) appropriately.

14.Does the bait protein necessarily need to be tested for self-activation?

The self-activation test needs to be carried out in the Y2H Gold strain. There are two main functions. One is to adjust the concentration of AbA or 3-AT according to the degree of self-activation of the bait protein; second, to detect whether the bait protein is toxic, if If the colony grows too slowly, the expressed protein may be toxic, and a low-copy plasmid is required to express the bait protein.

15.What effect does the concentration of AbA have on yeast screening?

ABA is used to screen positive clones, and the usual concentration of diimide is 100-200 ng/mL.Under the premise of normal negative and positive controls, if the number of positive clones screened out was too small, the ABA concentration was reduced to 100ng/mL.If too many positive clones were screened out, the concentration of ABA should be increased correspondingly under the premise of excluding self-activation.The effective concentration of ABA is also related to the amount of inoculation. For example, due to the excessive amount of inoculation, continuous colonies grow in the plate containing ABA, which makes ABA unable to achieve the effective effect.In general, 100 ng/mL, 150 ng/mL, 200ng/mL ABA is needed to screen about 2000 cells to inhibit the background expression of monohybrid recombinant yeast. If the number of cells is too large, ABA can not inhibit the background expression.

16.3-What is the selection of the screening concentration of AT? Is there a recommended concentration range for use?

Ideally, a 10-mm-concentration 3-AT plate will show little growth, and a 20-mm-concentration 3-AT plate will show no or little growth.If yeast was grown on a 3-AT plate AT a concentration of 80 mM, the self-activation activity was too high to be used for two-hybrid screening.

17. What if bait protein has self-activation activity and can independently activate the expression of reporter genes?

If the protein is a transcription factor with a transcriptional activation domain, the transcriptional activation domain needs to be removed.If it is not a transcription factor but still has a strong transcriptional activation activity, the activated region of bait protein needs to be removed for subsequent operation, but this operation may affect the interaction between proteins.

18.Does the size of bait protein affect the results of yeast two-hybrid?

Although there are successful cases from 8-750 amino acids reported in the literature, proteins with relatively large molecular weights may be misfolded in yeast. In actual work, bait proteins with relatively large molecular weights look for interacting proteins in yeast. The success rate is lower than that of bait proteins of medium molecular weight.

19. How to choose nuclear system or membrane system library for yeast two-hybrid?

If the bait protein is located on the membrane, the membrane system library is used.If the decoy protein was located in the nucleus, the nuclear system was selected.If the bait protein has a transmembrane region, it can be considered to remove the transmembrane region or select an intracellular region to use the nuclear system for screening, but there are certain risks.

20. What should I do if the expression of bait protein is not detected in yeast strains or if the expression is incomplete?

It may be that the OD value of yeast cells is too low and the number of cells is too small, leading to the low amount of bait protein, which is difficult to detect, so the liquid culture amount can be increased.Another reason may be that bait protein contains a large number of rare codon, which is difficult for yeast to translate. Therefore, it is suggested to optimize the amino acid sequence and synthesize a nucleotide that is composed of codon recognized by yeast cells and codes the same amino acid sequence, which is expected to greatly improve the expression of bait protein in yeast cells.

21. Can the positive clones screened on the four plates be directly sequenced with bacterial fluid or yeast plasmids?

No, because the yeast plasmid copy number is too low to reach the sequencing concentration, so we need to transfer the extracted plasmid to Escherichia coli culture and sequencing.

22. After the screened proteins were sequenced, why were many of the translated results from Aaa to Aaa shifted?

The purpose of the three-frame library is not to ensure no shuffling, but to ensure that every gene in the library has at least one unshuffling, that is, there is always the right protein in the target protein expressed in the library.Therefore, in the case of frameshift, it is necessary to analyze the sequencing peak map instead of only looking at the sequencing results, or there may be a deletion of a site on the peak map leading to sequence frameshift, which requires manual adjustment of the sequence.In theory, only unshuffled genes can be screened.

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