some basic questions about EGFP - (May/14/2010 )
1- after transfection of EGFP plasmids, it is recommended not to leave the cells under direct light because GFP protein will lose its intensity. I even tried this by myself and it is true. Does this affect the conformation of the protein? Is the GFP protein still detectable by western blot antibodies?
2- My friends transformed pEGFP-N2 with T7 promoters into Ecoli and after purification the protein glows in dark. I use the same plasmid but mine has a CMV promoter and I transfect cancer cells. So why do I see no fluorescence after protein purification in dark?
3- for how many passages after transfection the cells still glow?
-Curtis-
Curtis on May 14 2010, 12:12 PM said:
1- after transfection of EGFP plasmids, it is recommended not to leave the cells under direct light because GFP protein will lose its intensity. I even tried this by myself and it is true. Does this affect the conformation of the protein? Is the GFP protein still detectable by western blot antibodies?
2- My friends transformed pEGFP-N2 with T7 promoters into Ecoli and after purification the protein glows in dark. I use the same plasmid but mine has a CMV promoter and I transfect cancer cells. So why do I see no fluorescence after protein purification in dark?
3- for how many passages after transfection the cells still glow?
2- My friends transformed pEGFP-N2 with T7 promoters into Ecoli and after purification the protein glows in dark. I use the same plasmid but mine has a CMV promoter and I transfect cancer cells. So why do I see no fluorescence after protein purification in dark?
3- for how many passages after transfection the cells still glow?
1)http://micro.magnet.fsu.edu/primer/java/fluorescence/photobleaching/
Also, no, the photobleaching effect will not damage your ability to see GFP on a western.
2) It probably has to do with the massive quantity of protein you get from T7 in E. coli. You SHOULD, however, be able to see the GFP signal in the scope in your cells.
3) Very few. Without a selectable marker, the plasmid will be lost for energetic reasons. Producing GFP on a CMV promoter is very costly to the cell.
-Another Jake-
Another Jake on May 14 2010, 10:31 AM said:
Curtis on May 14 2010, 12:12 PM said:
1- after transfection of EGFP plasmids, it is recommended not to leave the cells under direct light because GFP protein will lose its intensity. I even tried this by myself and it is true. Does this affect the conformation of the protein? Is the GFP protein still detectable by western blot antibodies?
2- My friends transformed pEGFP-N2 with T7 promoters into Ecoli and after purification the protein glows in dark. I use the same plasmid but mine has a CMV promoter and I transfect cancer cells. So why do I see no fluorescence after protein purification in dark?
3- for how many passages after transfection the cells still glow?
2- My friends transformed pEGFP-N2 with T7 promoters into Ecoli and after purification the protein glows in dark. I use the same plasmid but mine has a CMV promoter and I transfect cancer cells. So why do I see no fluorescence after protein purification in dark?
3- for how many passages after transfection the cells still glow?
1)http://micro.magnet.fsu.edu/primer/java/fluorescence/photobleaching/
Also, no, the photobleaching effect will not damage your ability to see GFP on a western.
2) It probably has to do with the massive quantity of protein you get from T7 in E. coli. You SHOULD, however, be able to see the GFP signal in the scope in your cells.
3) Very few. Without a selectable marker, the plasmid will be lost for energetic reasons. Producing GFP on a CMV promoter is very costly to the cell.
Very good thank you for the answer. I too had the same questions and this helps. Now I have another question: Do the cells loose their fluorescence over a period of > 1 week, even if the cells are not split?
Thanks again.
-SciCell-
SciCell on May 14 2010, 03:13 PM said:
Another Jake on May 14 2010, 10:31 AM said:
Curtis on May 14 2010, 12:12 PM said:
1- after transfection of EGFP plasmids, it is recommended not to leave the cells under direct light because GFP protein will lose its intensity. I even tried this by myself and it is true. Does this affect the conformation of the protein? Is the GFP protein still detectable by western blot antibodies?
2- My friends transformed pEGFP-N2 with T7 promoters into Ecoli and after purification the protein glows in dark. I use the same plasmid but mine has a CMV promoter and I transfect cancer cells. So why do I see no fluorescence after protein purification in dark?
3- for how many passages after transfection the cells still glow?
2- My friends transformed pEGFP-N2 with T7 promoters into Ecoli and after purification the protein glows in dark. I use the same plasmid but mine has a CMV promoter and I transfect cancer cells. So why do I see no fluorescence after protein purification in dark?
3- for how many passages after transfection the cells still glow?
1)http://micro.magnet.fsu.edu/primer/java/fluorescence/photobleaching/
Also, no, the photobleaching effect will not damage your ability to see GFP on a western.
2) It probably has to do with the massive quantity of protein you get from T7 in E. coli. You SHOULD, however, be able to see the GFP signal in the scope in your cells.
3) Very few. Without a selectable marker, the plasmid will be lost for energetic reasons. Producing GFP on a CMV promoter is very costly to the cell.
Very good thank you for the answer. I too had the same questions and this helps. Now I have another question: Do the cells loose their fluorescence over a period of > 1 week, even if the cells are not split?
Thanks again.
That's really hard to say without just trying it empirically. Different cell-types will give you different results for sure. And you'd have to consider what you've got GFP tagged to as well. My bet is that in most cases, the results would be a high loss of signal over time.
-Another Jake-
Do you know how many KDa the GFP product of pEGFP-N2 is on western blot?
-Curtis-
Curtis on May 16 2010, 02:04 PM said:
Do you know how many KDa the GFP product of pEGFP-N2 is on western blot?
About 27
-Another Jake-