[Yeast] asking for information

Andrea Lo Ré via yeast%40net.bio.net (by andrealr82 from hotmail.com)
Tue Jul 14 13:00:01 EST 2009



Hello,Searching the net I found a response about liquid b-gal assays, and since you have vast experience on this issue I decided to write to you. I'm using pSV-Bgal for transfection normalization, so far I didn't get good results (at least not what I expected!), and I don't know if the problem is on the way used for detection. I'm doing assays with reporter systems and I must say I don't have much experience on this. I'm just starting my work (I'm a PhD student at Buenos Aires School of Medicine in Argentina), in fact I'm doing the first activity analysis in order to find the minimal promoter sequence of  a gene. I did some in silico research to learn more about the promoter, and got some "potential" promoters (using Mat Inspector). My first attempt was to take a 2000bp fragment upstream exon 1 by PCR on HeLa cells and start activity measurements. The gene of interest codes for a protein that plays a role in autophagy, and its expression is upregulated in different stress situations included starvation.
I cloned the sequence on pGL3 vector and checked activity after transfecting HeLa cells which were then treated under starvation conditions; as a control I used the pGL3 empty vector and for transfection control I used pSV40-Bgal.
So far I didn't got good activity results......I don't find consistent differences between treated and untreated cells (we suppose that in normal conditions the promoter is not going to be very active, or better said.....under starvation conditions we expect to have a great activation of the expression, and therefore have a strong luciferase signal)...so after dealing a lot trying to find the reason....I now blame it on everything!!! 
I first considered that maybe 2000bp is not enough and I should go further upstream the ATG.
Then I also wonder if the detection system is ok. I'm using a Firefly luc assay kit for pGL3 activity detection, and for Bgal I do a colorimetric measurement using ONPG at 420nm OD. I rinse the cells with passive buffer and use a sample for luminiscence and other for bgal. Every time I did it, samples took a long time to turn into yellow, and some of them even didn't changed at all, so results were so variable that I'm not sure about them..Do you have a standard protocol to measure B-gal activity? I also realised you normalized with protein concentration...In this case is it enough to have the b-gal value to normalize the luciferase results? or I should also take into account proteins concentration?I know you work with yeast but...=


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