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Date: | Tue, 26 May 2015 15:46:38 +0000 |
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Hi-
You might look at the Mann and Firtel papers, they looked at early gene expression. Because accumulation of a secreted factor called CMF regulates some early gene expression, you want to have the starved cells at a reasonably high density in suspension (ie more than 5 x 10exp6 cells/ml). Cells don't normally see much pulsing until ~6 hours, so pulsing should not be necessary.
best,
Richard Gomer
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From: DICTY [[log in to unmask]] on behalf of Kovarik, Michelle [[log in to unmask]]
Sent: Tuesday, May 26, 2015 10:24 AM
To: [log in to unmask]
Subject: [DICTY] development on solid vs in suspension
Hello All,
I am interested in studying the PI3K-PKB pathway in Dictyostelium during the early stages of the social cycle (e.g., first 6 hours). We'll be preparing lysates from the cells for biochemical experiments. We do not plan to do imaging experiments or to look at later stages of development or chemotaxis. I am wondering whether there are advantages to doing the development on a solid support (filter or agar) versus in suspension.
Is development on a filter always preferred for protein recovery? If we do the development in suspension, is it necessary to pulse in cAMP for the cell signaling to be normal? I know that to reach the later stages of development in suspension cAMP must be pulsed in, but I'm not sure whether this makes a difference at earlier times. I've been looking at the development page on the Dictybase website and have the Fey, et al 2007 Nature Protocols paper, but I am wondering if there are any papers comparing cellular signaling during the different development protocols.
I'd appreciate any pointers or references to relevant papers.
Thanks so much,
Michelle
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Michelle L. Kovarik
Department of Chemistry
Trinity College
300 Summit Street
Hartford, CT 06106
Clement Chemistry Bldg 129
[log in to unmask]<mailto:[log in to unmask]>
860-297-5275
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