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I find a website which has a lot of info http://www.copewithc...pe.cgi?key=WISH
According to this site, WISH is used to assay IFN alpha and gamma. So it seems that WISH is more sensitive to IFN than its original clone, HELA cell.
I found some WISH cells and hope I would get some good news...
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In Topic: Need help on STAT1-GFP nuclear translocation
31 January 2013 - 01:50 AM
In Topic: Need help on STAT1-GFP nuclear translocation
29 January 2013 - 08:41 AM
Typical IF protocol suggests 10min of cold methanol permeabilization. Have you tried 10min instead of 30min of methanol permeabilization? Would 10min be insuffisant?
When there is almost no IFN in serum, starving would not be necessary. But if there is a relative high concentration of IFN in serum, it could be problematic. Recently the quality of the serum in my lab is not so sure...
When there is almost no IFN in serum, starving would not be necessary. But if there is a relative high concentration of IFN in serum, it could be problematic. Recently the quality of the serum in my lab is not so sure...
In Topic: Need help on STAT1-GFP nuclear translocation
29 January 2013 - 06:56 AM
Should I starve the cells before IFN treatment in WB experiment? In publication it was not mentioned, hence I guess that it is not necessary. On the contrary, to stave the cells before IFN treatment seems necessary in IF experiment. Am I right?
In Topic: Need help on STAT1-GFP nuclear translocation
29 January 2013 - 05:53 AM
Hello! Doxorubicin,
I am not very experienced in intracellular staining.
To fix/permeabilize protein in nuclear, which method is better? Cold methanol at -20C for 10 min (I have red this method on several publications), or PFA 4%+ saponin 0.5% (this is rather for cytoplasmic protein)?
Which cell did you use for your IF experiment of phospho-stat1? I have use Hela and Hek293. I think that the 2 cell lines should have typical Jak/stat(1) signaling pathway.
Is WB more sensitive than IF? I find IF is pretty tricky.
I am not very experienced in intracellular staining.
To fix/permeabilize protein in nuclear, which method is better? Cold methanol at -20C for 10 min (I have red this method on several publications), or PFA 4%+ saponin 0.5% (this is rather for cytoplasmic protein)?
Which cell did you use for your IF experiment of phospho-stat1? I have use Hela and Hek293. I think that the 2 cell lines should have typical Jak/stat(1) signaling pathway.
Is WB more sensitive than IF? I find IF is pretty tricky.
In Topic: Need help on STAT1-GFP nuclear translocation
29 January 2013 - 05:25 AM
The phospho-stat1 translocation experiment was done with non-transfected Hela cell.
IFN should be OK. I have tried fresh stock of IFN and both IFN alpha and beta. I have worked with the aliquot of same IFN in other experiment that worked well. Hence the quality of IFN should be fine.
IFN should be OK. I have tried fresh stock of IFN and both IFN alpha and beta. I have worked with the aliquot of same IFN in other experiment that worked well. Hence the quality of IFN should be fine.
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