Hello,
We are performing quantitative yeast-two hybrid assays in the L40 strain with pDS-bait and pACT2-prey constructs. We are assessing dimerisation of one domain of a protein of interest, comparing the strength of a WT/WT v. WT/mutant(s) interaction. All the mutant constructs are in pDS while the WT, which remains constant throughout, is in both pDS and pACT2. We know from previously well-established data that the single amino acid substitutions should significantly decrease the strength of dimerisation. However, after several repeats, we continue to obtain inconsistent and highly variable results despite no obvious experimental errors. Technically, the assay works consistently ie upon all repeats yeast grows successfully and positive lacz assay results are observed. Moreover, all colonies picked for additional overnight growth develop to the correct optical density, with the quantitative CPRG assay consistently showing a measurable colour change. When analysing the results, however, we are unable to replicate previously published trends. There appears to be no significant difference between WT and mutant dimerisation, with most mutants even trending towards a potentially increased strength of interaction. We have sequenced and re-prepped all plasmid constructs, unfortunately to no avail. It would be immensely helpful to hear suggestions from experts re potential problems which we might have overlooked.
Current ideas:
1. Consistent cross-contamination of the DNA preps?
2. Quality of the yeast?
3. Plate reader issues?
4. Catching the yeast at the wrong growth phase?
Hi Simone,
Have you considered using 3-AT (3-amino-1,2,4-triazol). It is a competitive inhibitor. Starting concentrations in the plates could be 5-15-30-60 mM.
Even though you might also experience low reproducibility (because you add one more parameter), observing a concentration effect could help you to extract a trend.
Hope that helps,
Nico
https://www.researchgate.net/post/How_does_3-amino-1_2_4-triazole_3-AT_works_on_Yeast_two_hybrid_analysis
https://en.wikipedia.org/wiki/3-Amino-1,2,4-triazole
i think i will agree with Nicolas Brouilly , using 3-AT would help a lot as its a competitive inhibitor.
Dear Simone,
I am not familiar with your yeast strains and CPRG assay. However, you might want to consider the observations I made when performing ONPG assays with the LexA yeast two hybrid system.
(1) yeast cell lysis
I used to grow yeast liquid cultures in 96-deep-well plates and in order to obtain crude protein extract, I lysed the cells via feeze-thaw cycles. However, I ended up leaving out wells in the center of the plate, because not all wells were evenly frozen and thawed during the procedure. So depending on your lysis method, you might wanna double check if you evenly lyse the cells. You might even want to normalize against total protein in the extract instead of OD, to take differences in lysis efficiency into account.
(2)ONPG turnover
When adding chromogenic substrate (in my case ONPG) to the crude extracts, I observed ONPG turnover feedbacks into neighboring wells. I do not really have an explanation for that - but it has been my observation. The only way to circumvent that is to employ a randomized plate-setup with many repicates, and to rather use short incubation times before measuring absorbance, I think. As I said, I cannot tell which of the cleavage products should cause such an effect (maybe the o-nitrophenol is so volatile and affects beta-galactosidase activity???), and I cannot tell whether there are similar effects with CPRG.
Hope that helps. All the best for your troubleshooting!
(and of course the aformentioned 3-AT might also be a solution to your case)
As per my own experience, in quantitative assays the maximum variation is due to the uneven lysis of yeast cells and optical density measurement of yeast. To avoid uneven yeast lysis better to use the enzyme based protocols like Yeast beta-Galactosidase Assay Kit (Catalog number: 75768) from ThermoFisher Scientific. For optical density, take ODs between 0.3 to 0.5
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