yeast two hybridization - how to perform the X-Gal assay? (Jul/19/2004 )

Hello,

I am a pHD student and I just started working with the yeast two hybrid system to find new protein interactors with the protein I am studying.

I started by testing some known protein interactors in order to know if thechnically I am doing things well.

The problem is that I am able to get my transformed yeast growing in medium stringency selective media plates BUT I am not able to get positive blue colonies when I do the alpha-galactosidase assay.

My solution for the X-Gal filetr assy is composed by:
Z-buffer (pH 7.0) + 0.3mg/ml X-Gal (20mg/ml stock diluted in Dimethylformamide)+ 0.27% beta-mercaptoethanol

Z-buffer= 60mM Na2HPO4, 40mM NaH2PO4, 10mM KCl and 1mM MgSO4.

I don't know what is the purpose of using beta-mercaptoethanol in the solution. can someone tell me please? It is in the recipe given by Clontech but I don't know if I need to have it because other protocols don't use it.

I also don't know what is the best way and temperature to incubate the filters. Do you know if is better doing X-Gal at 30C, Room temperature or 37C?

thank you very much for your time,

Maria

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My understanding is that beta mercaptoethanol is often used to break disulfide bonds in proteins.

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Hi,

In our lab, x-alpha gal is usually added into the SD media plates and the colonies to be tested is streaked onto it.

If you are talking about filter lift off assay, I really hate the experiment as it requires you to dip the filter paper into liquid N2 and it becomes brittle and fragile. Is that what you guys do for alpha coz that is done for beta


Nevertheless, I dont really trust the results from X-gal assays.

regards,
tltan

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Thanks for the answer.
Actually you are right. I said I used alpha but I do the filter lift off using beta-galactosidase. The assay I did is working. As I had a positive control. two proteins known to be strong interactors and for those I got very bright blue colonies although with the protein I am interested in, the blue signal is extremely faint.

I started by trying X-Gal assay on filter because in Clontech protocol they say that this is more sensitive and less expensive than X-Gal in medium.

Can you see blue colonies only for very strong interactors or also for medium strenght interactions?

Thanks.

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Hello
I am deepti.I am also doing Yeast two hybrid.I want to know is there any protocol to use X-Gal for quantifying the interaction by doing B-gal activity assay?

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Hi Deepti,

I used the following protocol, is from Clontech. It works for strong intereractors.
if something is not clear I can write again explaning it better.
One important trick is that when you lift off filter should do it with a quick movement otherwise yeast cells don't come with filter.
Good luck.
Macedo


Reagents and Materials Required:
• Whatman #5 or VWR Grade 410 paper filters, sterile
Notes:
• 75-mm filters (e.g., VWR #28321-055) can be used with 100-mm plates; 125-mm filters (e.g., VWR #28321-113)
can be used with 150-mm plates
• Alternatively, 85- and 135-mm filters can be specially ordered from Whatman.
• Nitrocellulose filters also can be used, but they are prone to crack when frozen.
• Forceps for handling the filters
• Z buffer
• Z buffer/X-gal solution
• X-gal stock solution
• Liquid nitrogen
1. For best results use fresh colonies (i.e., grown at 30C for 2–4 days), 1–3 mm in diameter.
Notes:
• If only a few colonies are to be assayed, streak them (or spread them in small patches) directly onto master SD
selection agar plates. Incubate the plates at 30C for an additional 1–2 days, and then proceed with the
-galactosidase assay below.
• Use the SD selection medium appropriate for your system and plasmids. When testing LexA transformants, be
sure to use gal/raff induction medium.
2. Prepare Z buffer/X-gal solution as described in Appendix D.
3. For each plate of transformants to be assayed, presoak a sterile Whatman #5 or VWR grade
410 filter by placing it in 2.5–5 ml of Z buffer/X-gal solution in a clean 100- or 150-mm plate.
4. Using forceps, place a clean, dry filter over the surface of the plate of colonies to be assayed.
Gently rub the filter with the side of the forceps to help colonies cling to the filter.
5. Poke holes through the filter into the agar in three or more asymmetric locations to orient the
filter to the agar.
6. When the filter has been evenly wetted, carefully lift it off the agar plate with forceps and transfer
it (colonies facing up) to a pool of liquid nitrogen. Using the forceps, completely submerge the
filters for 10 sec.
Note: Liquid nitrogen should be handled with care; always wear thick gloves and goggles.
7. After the filter has frozen completely (~10 sec), remove it from the liquid nitrogen and allow it
to thaw at room temperature. (This freeze/thaw treatment is to permeabilizes the cells.)
8. Carefully place the filter, colony side up, on the presoaked filter (from Step C.3). Avoid trapping
air bubbles under or between the filters.
9. Incubate the filters at 30C (or room temperature) and check periodically for the appearance
of blue colonies.
Notes:
• The time it takes colonies producing -galactosidase to turn blue varies, typically from 30 min to 8 hr in a library
screening. Prolonged incubation (>8 hr) may give false positives.
• Yeast transformed with the -galactosidase positive control plasmid will turn blue within 20–30 min. Most yeast
reporter strains cotransformed with the positive controls for a two-hybrid interaction give a positive blue signal
within 60 min. CG-1945 cotransformed with the control plasmids may take an additional 30 min to develop. If the
controls do not behave as expected, check the reagents and repeat the assay.
10. Identify the -galactosidase-producing colonies by aligning the filter to the agar plate using the
orienting marks. Pick the corresponding positive colonies from the original plates to fresh
medium. If the entire colony was lifted onto the filter, incubate the original plate for 1–2 days
to regrow the colony.


For -galactosidase Filter Assays
• Z buffer
Na2HPO4
• 7H2O 16.1 g/L
NaH2PO4
• H2O 5.50 g/L
KCl 0.75 g/L
MgSO4 • 7H2O 0.246 g/L
Adjust to pH 7.0 and autoclave.
Can be stored at room temperature for up to 1 year.

• X-gal stock solution
Dissolve 5-bromo-4-chloro-3-indolyl--D-galactopyranoside (X-GAL; #8060-1) in
N,N-dimethylformamide (DMF) at a concentration of 20 mg/ml.
Store in the dark at –20C.

• Z buffer/X-gal solution
100 ml Z buffer
0.27 ml -mercaptoethanol (-ME; Sigma #M-6250)
1.67 ml X-gal stock solution

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