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yeast two hybridization


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#1 macedo

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Posted 19 July 2004 - 06:09 AM

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

#2 zienpiggie

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Posted 25 July 2004 - 04:43 PM

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

#3 tltan

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Posted 16 August 2004 - 06:26 AM

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

#4 macedo

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Posted 03 September 2004 - 12:37 AM

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

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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