Signal Transduction

Sugar

I use CCK8 kit to measure cell viability. Its fairly accurate and straightforward to use. Also non toxic. Can be used along with cytokines.

One thing that you would also need is a method to purify the protein from the cellular system out of which you would like to determine the protein's phosphosites. If it's possible try to do an immunoprecipitation of the endogenously expressed protein using a protein specific antibody. As this is not always possible, you can express a tagged version of your protein (preferably stable expression) and use the tag to IP purify. Once you have a good amount of IP purified protein (ideally a clear band by coomassie staining on SDS-PAG gel), you can send the protein to a mass spec facility. To identify phosphopeptides, the protein will be proteolytically cleaved. to get full sequence coverage, multiple proteases should be used with different cleavage properties (trypsin, thermolysin etc). I hope this helps.

@Daphne- prokaryotic and eukaryotic DNA methylation patterns are quite different. Do you know of any publications that show that prokaryotic methylation affected the behavior of a luciferase reporter? This would be useful information to catalog.

Does ti works well with S. cerevisiae? Do you know the exact reference or name? thanks

Dear Mr. Tayaranian, you are right to assume that mTOR leads to autophagy. There are two articles which can increase your understanding about the subject. They are: Cell death in the nervous system - Dale E. Bredesen, Rammohan V. Rao & Patrick Mehlen. NATURE|Vol 443|19 October 2006|doi:10.1038/nature05293 and Autophagy and Cell Death: No Longer at Odds - Andreas Bergmann Cell 131, December 14, 2007

Why do you think that there will be a big difference between human and mouse? I think if your drugs are working for mouse macrophages it should also work out for human macrophages. you just should find the right concentration of your inhibitor. The experimental question sounds good (but I do not have experience with leishmania; I am working with Listeria -> NFkB also helps to fight against Listeria)

Try Kinase-Glo ® Luminescent Kinase Assay , Best, Thiru

Ask for ESA detectors. the hpls pump could be any brand

We have successfully utilized lucifer-yellow in optic nerve sections in vitro, then performed imaging with confocal microsc.

Yes, I have studied cAMP assays and also developed high-throughput screening. Amersham, Assay Design, Millipore, Luciferase, and CNG (cyclicnucleotide gated ion channels). I found Millipore is a simple, cheap and short time assay and working wonderful. Luci takes more time and require stronger hits and is not a robust for high-throughput screening. CNG assay is good for high-throughput screening. Depending on number of samples you can design which one would be suitable for your experiments. Thanks, and goodluck. Syed

In addition to Fura-2AM , you can also use cytoplasmic targeted Ca2+ probes e.g cameleon based probe

Your answer has been very helpful - thank you Sylviane!

PyRosetta does this pretty well. You'll need some (not much) experience with coding. The tutorial describes how to do a first approximation (or back of the napkin approach). Tutorial https://docs.google.com/viewer?a=v&pid=sites&srcid=ZGVmYXVsdGRvbWFpbnxweXJvc2V0dGF8Z3g6NzY0ZmY3YWNkYWVkNWQ2Mw PyRosetta is here: http://www.pyrosetta.org/

Hi Dr. Helmut, Following link shows the article where I used static in macrophages. http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0027685 Thanks.

I agree, I would not consider mTOR Ser2448 autophosphorylation to be a reliable indicator of mTOR inhibitor activity as this site has been shown in some papers to be mTOR-dependent and in others non-mTOR-dependent, which could ultimately confuse your results. In my personal experience, both the immediate downstream kinase and its kinase substrate (p70 S6 kinase and S6, respectively) will give consistent and reliable readouts of mTORC1 activity; in this regard, pS6K (Thr389) and pS6 (Ser235/236) are the two best sites to look at although certain phospho-sites on 4E-BP1 can also be utilized. If you are screening to inhibit mTOR kinase activity in general, mTORC2 activity can be actively monitored by looking at the pAkt (Ser473) site. Positive inhibitor controls for mTORC1 and mTORC1 + mTORC2 activity would be rapamycin (2nM overnight or 20nM for 5 - 7h in most cell lines) and WYE-132, respectively. Of note is the observation of a rapamycin-mediated feedback mechanism (via IRS-1) that results in the activation of pAkt (Ser473) in some cell types upon prolonged rapamycin exposure, which should be taken into consideration in your assays; http://sabatinilab.wi.mit.edu/Sabatini%20papers/mTORC2_rapa-MCell-2006.pdf With respect to fluorescence-mediated monitoring of mTOR inhibition you might want to look into the LANCE Ultra ULight p70 S6K and 4E-BP1 kits, which are TR-FRET-based kinase assays; http://www.perkinelmer.com/Catalog/Category/ID/LANCE%20Ultra%20Acceptor%20dye%20conjugates%20Kinase%20assay%20ULightlabeled%20substrate%20reagents Alternative TR-FRET kinase assay kits include the LanthaScreen kits by Invitrogen; http://tools.invitrogen.com/content/sfs/posters/0407-SBS-mTOR-Protein-and-Assay.pdf Other potential screening options are the K-LISA kits from EMD Biosciences; http://www.emdmillipore.com/life-science-research/k-lisa-mtor-activity-kit/EMD_BIO-CBA055/p_uuid?ProductID=056b.s1Oh64AAAEjskBHkR1L Cell Signaling also have the fluorescent-based PathScan assays, which would allow you to look at both mTORC1 and mTORC2 activity, via p70 S6K (Thr389) and pAkt (Ser473) respectively; http://www.cellsignal.com/products/7744.html However, given the expense and broader pathway coverage that these PathScan assays offer I would probably use this as a final step to look at off-target effects of mTOR inhibitors based on output generated from more focused, cost-effective assays as listed above. I hope this information helps and good luck with your research! Cheers, Jonathan

Dear Maria Which kinase you want to study? 1- Usually you can detect the phosphorylation of kinase subestrates. 2- By immunopercipitation western blot you can do kinase assay.

Dear Felix, Thank you . A very useful list of publications. Best regards Saranga