October seminars/Intracellular Training Course

barteneva@cbr.med.harvard.edu barteneva at cbr.med.harvard.edu
Thu Sep 27 13:12:57 EDT 2007

Dear Collegaues:

We have 2 interesting seminars in October; Georgyi Loss from Promega, October 2nd
and Robert Balderas from BD Biosciences, October 30th.

Also, we are hosting Intracellular Flow Course (with Flocyte Inc) as a satellite to NEC
annual meeting on 17th October (requires registration/payment on www.flocyte.com, please,
contact Sue DeMaggio)

Upcoming Seminar: next Tuesday, 2nd October, 3 pm, Latham Library, Immune Disease
Institute, HMS, 200 Longwood Ave GEORGYI V. LOS , Imaging Group Leader, PROMEGA 
HaloTag ®: Novel Protein Labeling Technology for Cell Imaging and Protein Analysis.

Development of novel reporter systems for live cell imaging and protein analysis is
essential for further advancement of life science research and drug discovery. Recently
we have described a technology for site-specific covalent tethering of synthetic ligands
to a reporting protein, HaloTag ®, in living cells and in vitro. The reporter protein is
a genetically engineered catalytically inactive derivative of hydrolase, has globular
structure and remains monomeric even at high concentrations. The ligands are small
chemical tags capable of carrying a variety of functionalities, such as fluorescent
labels, affinity handles, or attachments to a solid phase. The covalent bond forms
rapidly under general physiological conditions, is highly specific, and essentially
irreversible. Fusion of the HaloTag ® protein to cytosolic, membrane anchored, or
transmembrane proteins does not interfere with native protein function. The open
architecture of the technology ensures interchangeability of
ligands, thereby facilitating a variety of functional studies (including imaging at
different wavelengths and temporal or spatial separation of protein pools) without
requiring changes to the underlying genetic construct. The stability of the bond between
the HaloTag ® protein and HaloTag ® ligands allows imaging of live cells during long
periods of time, imaging of fixed cells, stimulated emission depletion (STED) microscopy,
and multiplexing with different cell/protein analysis techniques. The HaloTag ® protein
retains activity upon fixation making possible covalent tethering of functional groups to
fusion proteins in fixed cells. Attachment of the HaloTag ® ligands to different surfaces
allows oriented, highly specific covalent immobilization of HaloTag ® fusion proteins.
These surfaces can be used for detection of protein-protein and protein: DNA

October 30th, 10 am, Cannon Room, HMS tetrangele, Robert Balderas, Vice-president of
Research&Development, BD Biosciences; Novel Flow Cytometric Techniques for Analysis of
Protein Phosphorylation and Signaling Networks

Intracellular assays of signaling systems have been limited by the
inability to correlate functional subsets of cells in complex populations
on the basis of active protein states within the native context of the
cell. We demonstrate the ability to simultaneously monitor active protein states via
phospho-epitope recognition in subpopulations of complex cell populations by
multiparameter flow-cytometric analysis. Multi-dimensional assessment of active protein
states, in combination with surface marker and other flow cytometric detectable
parameters (ie, cytokines, apoptosis), can provide functional assessment on a single cell
level that may have utility in clinical diagnostics and/or disease progression.
Furthermore, the ability to profile both activating and inhibiting conditions of multiple
protein states simultaneously within the cell in a rapid and parallel manner may be
extended to pharmaceutical screening of compounds. Detailed
examples for the utililty of assessing the signaling pathways using flow
cytometry in  T/B cell and Cancer Models will be provided.

Dr Natasha Barteneva
Immune Disease Institute
Director,Flow and Imaging Cytometry Core,
Harvard Medical School
200 Longwood Ave, #239(Armenise Building)
Boston 02115, MA
phone: 617-278-3037
fax: 617-278-3030
e-mail: barteneva at cbr.med.harvard.edu

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