deer fellow flow fellows, I pose two questions, and await the word ambulance.... 1. I understand there are ?antibodies? against phosphorylated tyrosine kinase proteins. We infact have used one from Sigma PT66 in recent past, found it to be specific and quite useful. However there is a dilemma... Since the antibody stains all tyrosine kinase proteins (TKp) we are looking at changes in whole phosphorylation within a population of cells eg monocytes. Applying this to a question such as "is there an overall increase in phosphorylation of TKp upon stimulation of phagocytes with eg. beads? and does the extent of this phosphorylation alter or diminish with infection eg HIV" I would like to make this study even more specific by detection of individual phosphorylated proteins rather than a whole spectrum. I noticed there are different extents of phosphorylation pending on isolation. ie cells elutriated give strongs primary signals whereras adhered cells being already activated give weaker secondary signals. Are there different proteins acting with different stimuli? By using methods such as coating beads with complement or IgG we get look at two mechanisms of phagocytosis, them being complement and Fc mediated.. questions such as the overall fluorescence (looking at mean fluorescence on a univariate or single peak data) may not change much (insignificantly?) when comparing the two conditions but different proteins may be phosphorylated (my theory being a constant phosphorylation and dephosphorylation within the cell of different proteins may give me very similar data, but a totally different group of proteins may be phosphorylated). So is there a commercial product that is available to hone in on specific TKp's eg. srk or hck or pyk or any, I know there are products to be used with western blotting, but has anyone applies these to flow?? Or are there some available specifically for flow... or am I looking for a needle in a haystack. 2. Secondly I am very much interested in hearing from anyone whom works with HIV and flow. We have used an anti p24 antibody in the past, however this does not single out individual infected cell populations as whole peak shifts are observed... meaning what I get is ALL cells infected to varying degrees... I observe this in infected macrophages. What I'd like to do is single out (get bimodal distribution +/- populations) with clear distinction between infected and uninfected macs or any other cells for that matter... is anyone out there in flowland already working with this or is there a product which will enable me to achieve this... has anyone cloned GFP into HIV yet?? or better still without altering HIV has anyone directed an anti HIV RNA GFP-vector? is there a better marker than p24?? all your answers are much appreciated in advance and fanx for reading the email... sorry for the long windedness.. ekat erac lla Geza Paukovics - Flow Laboratory - AIDS Pathogenesis Research Unit Macfarlane Burnet Centre for Medical Research .***. .***. .** PO Box 254 _--_|\ * | | | * * | | Fairfield, VIC 3078 / \ * * | | | * * | | | AUSTRALIA. \_.--. _ / * * | | | * | | | * ph. (+61 3) 9282 2132, O '***' '***' FAX (+61 3) 9282 2100
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